“Victor Babes” National Institute of Pathology
SELF-ASSESSMENT REPORT
2007-2011
Table of Contents
1. Functional administrative structure diagram
2. General activity report
3. Activity report by team
4. Representative project
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1. Functional administrative structure diagram
Management team: - General director: Acad. Laurentiu M. POPESCU - Scientific director: CSI Dr. Mihail Eugen HINESCU - Director: CSI Dr. Bogdan Ovidiu POPESCU - Economic director: Ec. Mariana GEORGESCU Decision-making structures: Administrative Council (9 members), Scientific Council (13 members), Board of directors (4 members). Departments’ organization
PATHOLOGY Dr. Carmen Ardeleanu
IMMUNOLOGY Dr. Cornel Ursaciuc
BIOLOGY Dr. Mihail Hinescu
Histopathology, immunohistochemistry and molecular diagnosis
Prof. Dr. Carmen Ardeleanu
Immunobiology
Dr. Monica Neagu
Medical genetics
Dr. Aurora Arghir
Ultrastructural pathology
Dr. Mihaela Gherghiceanu
Immunopathology
Dan Ciotaru
Cellular medicine
Dr. Mircea Leabu
Biochemistry
Dr. Cristiana Tanase
Animal husbandry
Bogdan Marinescu
Molecular medicine
Dr. Bogdan Popescu
Diagnosis center
Georgeta Butur
Radiobiology
Dr. Gina Manda
Since 2005 the Institute is certified SR EN ISO/CEI 9001:2008 for research activity and medical services. Several laboratories received national accreditation: Histopathology and immunohistochemistry laboratory (accreditation SR EN ISO/CEI 15189:2007), Biochemistry laboratory (accreditation SR EN ISO/CEI 15189:2007), Animal Husbandry unit (accreditation SR EN ISO/CEI 17 025: 2005), Nuclear unit (national authorization by CNCAN).
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2. General activity report Founded in 1887, “Victor Babes” National Institute of Pathology from Bucharest is the first institute of biomedical research in Romania. Still at the forefront of fundamental research, the Institute is today a center of reference for human disease diagnosis and monitoring.
Mission
Victor Babes Institute of Pathology mission is to conduct cutting edge research in the field of molecular and cellular medicine for the knowledge-based scientific progress in the benefit of society.
We are using all organizational resources to address major societary needs in the area of health and to provide scientifically sound instruments and solutions in the benefit of patients and health professionals. The institute correlates the identified needs at national level with the scientific and health challenges at European level, thus providing the most effective ways of access to state of the art knowledge / solutions and acting as a scientific connection with health and research entities in Europe. The institute provides support for strategic planning and decision at national level for policy makers in the field of biomedical research and healthcare.
The institute’s mission is to expand the knowledge in biomedical and associated sciences by conducting and supporting research, development, education / training and high-quality medical services. The institute’s mission constructively influences the quality of life and healthcare services at national level.
The institute is committed to increase the international visibility of Romanian research in the field of cellular and molecular medicine.
Sharing efforts with partner medical institutions (in an attempt to build a local translational research community), the institute is responding with up-to-date solutions to major human health issues in cancer, (neuro)degenerative diseases, immune disorders, nephropathology and cardiovascular diseases. The institute, organized in three main research departments (pathology, immunology, biology) is using state-of the-art methodology to develop innovative diagnostic tools and personalized medicine strategies.
The advantage of having infrastructure and experts in different disciplines is that we can adjust our projects along the way, depending on the priorities related to patient needs and in conjunction to the European scientific trends. It is our commitment to be integrated in large scale multidisciplinary projects in biomedical science, aiming at improving health and the quality of life.
Research focus: Multidisciplinary research in the field of cellular and molecular medicine, developed by 10 research teams
1. Telocytes: telocytes characterization; telocytes-stem cells tandem; telocytes in regenerative medicine; in vitro and in vivo functional studies on telocytes
2. Surgical and molecular pathology: cell signaling pathways in malignant epithelial tumors; molecular bases of therapy modulation in malignant tumors; genotypic profiles variability in cancer
3. Translational research in cancer: therapeutic targets in malignant tumors; prognostic and predictive biomarkers in epithelial malignant tumors; molecular identification of etiologic factors in infections and associated tumors
4. Ultrastructural pathology: basic research in fundamental mechanisms of cardiac regeneration - from stem cell to heart tissue; 3D electron tomography of the caveolar microdomains in smooth muscle cells; cellular and molecular mechanisms involved in glomerular pathology; cell ultrastructure investigation
5. Proteomic biomarkers: proteomics technologies for biomarkers discovery in cancer; proteomics biomarkers in pharmacological research; proteomics in the evaluation of environmental risks for human health
6. Immunomodulation-immunodiagnosis: tumor immunology; biomarkers in autoimmune diseases; cytokines and immunomodulation; innovative immunotherapies
7. Genomics and genetic diagnosis: genetics of neuropsychiatric disorders; genetic/epigenetic and genomic biomarkers relevant for cancer onset and progression
8. Neurosciences: trophic factor receptors expression in central and peripheral nervous system; tight junction proteins in brain and peripheral nerves; neurodegeneration models relevant to Alzheimer and Parkinson diseases; neuromuscular pathology
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9. Drug development and toxicology: pathologic mechanisms and drug targets in cardiovascular diseases and rheumatoid arthritis; drug development - biological in vitro and in vivo screening; immunotoxicology; radiobiology
10. Assay development and alternative testing: immune-based assay development for bacterial/viral infections; cell-based assays development for drug assessment and nanomedicine.
Collaborations
- National collaborations with partners having complementary expertise and infrastructure - International collaborations with Max Planck Institute, Graz University, University of Tuebingen,
Universite Catholique de Louvain, Ludwig Cancer Institute, University of Goteborg, University of Turin, University of Medicine Florence, University of Athens, Cyprus Institute of Genetics and Neurology, Center of Cardiovascular Research Aachen, Saint George’s University of London, Descartes University of Paris, Hospital Cochin, Chinese Academy of Medical Sciences etc.
Project-based research Research excellence in the field of life sciences is sustained by a broad array of research projects: International projects: 4 bilateral projects with France, China and Cyprus, 1 NATO Science for Peace project, 1 MNT-ERA NET project, 1 FP7 – People project, 1 project in the EU Education and Culture-Lifelong Learning Program. Research structural funds: 2 POSCCE projects with foreign coordinator (Priority axes 2 – Competitivity by research, technological development and innovation)
o Proteomics technologies for cancer biomarkers discovery (coordinator Prof. S. Constantinescu) o Implementation of molecular tissue assays for cancer in Romania. State-of-the-art research
focused on personalized oncology (coordinator Prof. G. Bussolati) Projects financed by the national research programs CEEX (Health, Biotech, Matnantech, Infosoc), CNCSIS, PNII Partnerships and Capacities = 125; 21 projects were coordinated by INCD “Victor Babes” and in 104 projects the institute participated as partner
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2007 2008 2009 2010 2011
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500000
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2500000
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3500000
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4500000
5000000
bu
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euro
s)number of projects
budget
The significant cut-offs in number of granted projects and budgets are explained by lack of national research competition calls in 2009 and 2010, and a dramatic decrease in research public funding as a result of economic slow-down. The 2010-2011 up-trends reflect accessing of European structural funds. Getting alternative financial support in the frame of Sectorial Operational Program for Increased Competitiveness is an important advantage for research institutes. However, this opportunity was not a long term option, since the current call topics do not fit the institute’s expertise/eligibility. Projects financed by the European Social Fund: 3 projects focused on the training of personnel from the national health system in the field of state-of-the-art biomedical techniques, aiming to implement new diagnosis tools in clinical laboratories. Infrastructure development projects: 1) Advanced infrastructure for molecular cytogenetic research; 2) Upgrading of a biobank for tumor cells and nucleic acids by attaching an immunogenomics laboratory for molecular screening in cancer; 3) Upgrade of research infrastructure for laboratory animals in INCD "Victor Babes"; 4) Makeup of the most competitive laboratory in Romania for living cell direct study under microscope in an incubator Project for defining strategic priorities: “Cell therapy in regenerative medicine development. Strategic priorities” – STRATEC, funded by the National Authority for Scientific Research. Through this project
EU Structural Funds
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2007 2008 2009 2010 2011
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2007 2008 2009 2010 2011
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rAIS
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rAIS
citations
the institute offered its expertise to the main research policy maker in Romania and our researchers gained new insights in the field of cell therapies, paving the way to future research directions.
Major achievements
Publications
Publications in ISI journals with non-zero rAIS = 99
- total rAIS = 154,15918 - rAIS/researcher = 2,03 - total number of citations = 561 - mean citations number/researcher = 7,38
The institute’s research activity became significantly more visible at international level during 2008-2011, both as publications and citations number in ISI ranked journals. Considering the number of contributing researchers (76), it is obvious that the objective of the institute’s strategy to enhance international visibility was reached.
Publications in ISI journals with zero rAIS = 48 Other publications
77 papers published in non-ISI journals
9 books (1 published by Elsevier) and 20 book chapters (7 published by international publishing houses)
Patents - 4 registered patents 1) Tetra-sulphonated porphyrin application for producing a dermatologic
therapy – photosensitizer; 2) Tetrapirolic compound asymmetrically substituted – synthesis and biological evaluation; 3) Equipment and procedure for microwave irradiation in in vitro models with concomitant registration of biological behavior in a fluorescence microscope; 4) Method of obtaining yeast bioproduct enriched with chrome; 2 submitted patents.
The patent “Tetra-sulphonated porphyrin application for producing a dermatologic therapy – photosensitizer” received Gold medal at Brussels Innova 2008, Special Prize of Rudy Demotte, Minister President of the Walloon Government, Gold medal at The 37th International Exhibition of Inventions of Geneva 2009 and Special Prize of the Ministry of Education of Russia, 2009; Gold medal at The International Fair for Innovation, Moscow, 2009.
The project proposals of the Institute at the 2011 Call “Partnerships – Collaborative projects of applied research” reflect our commitment to develop applied research in consortia with other public and private R&D institutions, resulting in patents and publications.
Research results translated to the Diagnosis Center: New immunohistochemical algorithms for the diagnosis of cancer; In situ hybridization for Epstein Barr virus in undifferentiated carcinoma of nasopharynx and Hodgkin lymphoma; Fluorescent (FISH) and chromogen (CISH) in situ hybridization for Her-2/neu gene amplification in breast and gastric carcinoma; FISH for bcl 2 translocation in follicular malignant lymphoma; FISH for gene fusion in Ewing sarcoma; FISH for ALK gene mutation in anaplastic malignant lymphoma and in bronchopulmonary adenocarcinoma; Molecular
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2007 2008 2009 2010 2011
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R&D/ADM staff ratio
y = 0,427x + 2,2871
R2 = 0,9917
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3,5
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4,5
2008 2009 2010 2011
number of emplyees
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200
400
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1.000
1.200
2007 2008 2009 2010 2011
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detection of K-ras gene mutation in colon carcinoma; c-kit gene mutation in GISTs; EGFR gene mutation in lung adenocarcinoma (in preparation). Staff: 76 researchers, out of which 21 senior researchers and 19 PhD students, along with 39 technicians and 34 NRDS personnel (situation at 15 December 2011), with a mean age of 44 years, represent a critical mass for self-sustaining and further growth in biomedical research. We are committed to recruit young scientists and to offer them adequate support to develop competitive research. The stability of R&D personnel indicates that the institute assured appropriate conditions for research and career development.
Major achievements of the institute’s human resources policy
- Recruitment as project coordinators of foreign scientists with outstanding scientific visibility: Prof. Stefan Constantinescu and Prof. Gianni Bussolati
- Recruitment of young researchers with foreign experience in prestigious research institutions and universities: Valeriu Cismasiu – specialization in stem cells: post-doctoral fellow at Lund University, Stem Cell Center, Sweden (2007-2009) and EMBL Mouse Biology Unit, Monterotondo, Italy; visiting scientist at Weatherall Institute of Molecular Medicine, Oxford, UK (2008-2009). Andreea Tutulan-Cunita – specialization in molecular biology and genetics; master degree (2000-2002) and PhD (2002-2005) at Hiroshima University, Japan; postdoctoral fellowship (2005-2006) at Manchester University, UK. Sevinci Pop – specialization in transcription activation, DNA-protein interaction, chromatin structure and function, gene expression; doctoral fellowship (2000-2002) and postdoctoral fellowship (2003-2008), University of Illinois at Urbana-Champaign, USA,. Georgeta Cardos – doctoral fellowship (2004-2008) at Hamburg University, Germany
- Training of our researchers in prestigious research laboratories
mean number of employees
130
135
140
145
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155
160
165
170
175
2007 2008 2009 2010 2011
number of employees
evolution of employees number by category
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CSI CSII CSIII CS AS TS AUX ADM
number of employees
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2011
There is an obvious increase trend in the R&D/ADM personnel ratio during the last 4 years, proving an appropriate human resources policy according to the main activity of the institute (research).
The personnel structure reflects the needs of the institute for project development and for reaching excellence in life sciences research.
Infrastructure upgrading was one of the main priorities of our strategic plan for 2007 - 2011. Funds were obtained from 4 specific projects financed by the national Capacities Program and from research projects. Investments were focused on developing cytometry, imagistic, genomics and proteomics research units, a biobank for tumor cells and nucleic acids and the animal husbandry.
* Taking together our achievements in the last 4 years, we conclude that “Victor Babes” Institute of Pathology has a leading position in life science and biomedical research in Romania.
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3. Activity reports by team
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TEAM 1 - TELOCYTE - A NEW TYPE OF INTERSTITIAL CELL
Team leader: Laurentiu M. Popescu
Senior Researchers: Mihail Eugen Hinescu; Eugen Mandache
PostDoc researchers: Mihaela Gherghiceanu; Bogdan Ovidiu Popescu; Sanda Cretoiu
Laura Cristina Suciu; Valeriu Bogdan Cismasiu; Bogdan Gabriel Marinescu
PhD students: Catalin Gabriel Manole; Dragos Cretoiu; Mihnea Ioan Nicolescu
Technicians: Marin Teodor Regalia; Maria Dumitrescu; Rodica Stanca; Petrica Musat
L.M. Popescu, MD, PhD, Dr. h.c.mult., is currently Head of the National Institute of Pathology, Bucharest,
Romania. He is member of the National Academy of Sciences and of the Academy of Medical Sciences.
Recently, he became President Elect of the Federation of European Academies of Medicine, and of the
International Society for Adaptive Medicine. He published over 100 scientific articles in international peer-
review journals and is cited more than 1500 times. He has a Hirsch Index of about 30. Professor Popescu
is Editor-in-Chief (and founder) of the Journal of Cellular and Molecular Medicine (Wiley/Blackwell), with a
5-year IF of 5. He is credited with the discovery of Telocytes.
Recently, our team discovered a new cell type in humans and mammals. We called recently (2010) these
cells Telocytes, replacing the term previously used by us – Interstitial Cajal-like Cells (acronym: ICLC).
This discovery is cited in more than 100 scientific papers, and very recently we are trying to impose the
concept of “Telocytes / Stem-Cells Tandem” existing and working in the so-called Stem Cell Niches. The
Telocyte concept is already adopted by many scientists: e.g. Eyden et al. – UK; Faussone-Pellegrini &
Bani – Italy; Gittenberger-de Groot et al. – The Netherlands; Klumpp et al – Germany; Kostin – Germany;
Polykandriotis – Greece; Zhou et al – China; Cantarero et al - Spain; Gard & Asirvatham – USA; Gevaert
et al. – Belgium; Marban et al – USA; Limana et al – Italy; Radenkovic – Serbia; Rupp et al. – Germany;
Russell – USA; Tommila – Finland, etc.
Major research topic is the connective tissue (CT) cellular and molecular biology with emphasis on
telocytes involvement in tissue physiology and pathology. CT represents the essential microenvironment
to coordinate body structure and function hosting and joining together three major systems (the
circulatory, nervous and immune systems) and integrating all others tissues and organs. More than
200,000 publications showed that CT is involved in organ development, renewing, repair, regeneration,
and tumour development. Recently, new types of cells (e.g. resident mesenchymal stem cells, resident
stem cells and telocytes) have been described but specific markers for the majority of the CT cells are still
missing. Their detection and discrimination in situ is highly biased by their almost exclusive
characterisation in vitro.
Major questions concerning CT biology must be addressed:
How many distinct cell types reside in the adult CT?
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Are these cells tissue specific?
Do stem cells need a specific CT microenvironment to survive and differentiate?
How does the CT network change in specific pathological conditions?
We plan to perform a systematic inventory of the old and the new cell types of the CT, their molecular
profiling, tissue distribution and interactions with emphasis on fibroblasts, fibroblast-like cells, telocytes,
and mesenchymal stem cells.
Future research plans are to:
define specific phenotypic, genetic and functional markers for particular CT cells and to assess
their tissue specificity;
identify novel cellular, molecular and signalling networks involved in CT physiology and
remodelling;
re-evaluate homo/heterocellular and cell-to-matrix communication;
investigate cellular aspects of tumour-stroma interaction.
Our approach involves structural/ genomic/ transcriptomic/ proteomic/ functional assays using in situ/ in
vitro/ in vivo models to define the CT ‘connectomics’. CONNECT research could offer new ways for
regenerative medicine (e.g. poly cellular treatment instead of stem cell approach only) as well as anti-
tumour therapies for stromal tumours.
Main expected outcomes of our research:
1) Better characterized resident CT cells (e.g. fibroblasts, mesenchymal stem cells, telocytes) and their
microenvironment
2) Endorsing markers for CT cells recognition in situ
3) Increasing knowledge about cellular and molecular mechanisms of tissue renewal
4) Better characterization of telocyte - the cell described in preliminary studies by our group
Existing equipment and facilities:
- electron microscopy unit (ultrastructural analysis, cellular tomography, array tomography):
Transmission electron microscope Morgagni 268 FEI, 100 kV; Olympus MegaView CCD; Electron
microscope Tecnai G2 BioTwin Spirit FEI, 120 kV, single tilt holder; Olympus MegaViewG2 CCD;
UV/Cryo-chamber EMS; LKB and RMC XL ultra-microtomes; diamond knifes, ovens, etc.
- identification and localization of molecules by immunofluorescence: laser scanning microscope
Nikon; microscopes Nikon E 600 with UV, CCD; 2 Nikon 200; Leica cryotom; immunostainer, paraffin
embedding station; biobank for tissues, cells and nucleic acids; deep freezing unit; Leica microtome.
- light microscopy facility: motorized AxioZ1 Zeiss microscope with light bright field and
epifluorescence, equipped with high resolution monochrome cooled CCD camera and image processing
software.
- cell culture facilities: Laminar flow hoods; CO2 Incubators; Centrifuges; Phase contrast microscope,
Sterilization unit, Ultra pure water system.
- cytometry unit: fluorescence and confocal microscopes, flow-cytometer Becton-Dickinson
FACSCalibur
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- videomicroscopy: Biostation IM (Nikon Corp., Japan).
- cell layer impedance measurement: xCELLigence (Roche Diagnostics, Germany).
- microarray facility: Agilent DNA Microarray Scanner (G2565CA) with SureScan High-Resolution
Technology with Agilent Scan Control Software and Genomic Workbench; Agilent BioAnalyzer and
hybridization oven.
- molecular genetics facility: Corbett Palm Cycler PCR, Corbett Real Time PCR;
- nucleic acid isolation and manipulation facilities: chemical hoods, water baths, centrifuges,
spectrophotometer, transilluminator, freezers, ultrafreezers.
- microRNA qPCR: NanoDrop ND-1000 Spectrophotometer; QuantiMir RT -System Biosciences; iCycler
system and software- Bio-Rad.
- proteomics: SELDI-TOF mass spectrometry system, Luminex-xMAP; 2D and 2D-DIGE; software –
PDQuest; protein microarray platform.
- animal Models Core Unit (SR EN ISO 17025:2005)
- image processing: photographic laboratory; 4 Dell computers, 1 Siemens; Wacom Intuos 3 pen tablet;
software for image analysis: Axiovision Zeiss; ImageJ -NIH, iTEM FEICo, Explore3D FEICo, Amira.
International collaboration:
Prof. Maria-Simonetta Faussone-Pellegrini and Prof. Daniele Bani from Department of
Anatomy, Histology and Forensic Medicine, University of Florence, Italy
Prof. Ofer Binah from the Department of Physiology; Ruth and Bruce Rappaport Faculty of
Medicine, Technion Israel Institute of Technology, Haifa, Israel
Prof. Sawa Kostin from Max-Planck Institute for Heart and Lung Research, Franz Groedel
Institute, Bad Nauheim, Germany
Prof. Shengshou Hu from the Center for Cardiovascular Regenerative Medicine, Peking Union
Medical College, Chinese Academy of Medical Sciences, Beijing, China
Prof. Changyong Wang from the Tissue Engineering Research Center, Academy of Military
Medical Sciences, Beijing, China
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TEAM 2 - SURGICAL AND MOLECULAR PATHOLOGY Team coordinator: CS1. Prof. Dr. Carmen Ardeleanu, Head of Pathology Department Mission: Obtaining new tissue biomarkers regarding the structure and molecular profiles of pathological processes (tumors, inflammations etc.) Research focus:
Molecular biology of malignant tumors focusing on inter- and intracellular signaling.: exploring signaling pathways molecular factors
o development and implementation of high throughput methodologies using in vitro assays with particular end-points, for identifying and characterization of new biomarkers of cell signaling in tumors
o partnership with other research teams from clinical oncology, surgical clinics, for testing and promoting new diagnostic and therapeutic tools potentially efficient in cancer
Identifying new genes implication in tumor progression by means of genotypic and phenotypic profiles
o implementation of advanced molecular methods for characterizing the genetic variability of malignant tumors
Developing and extending standardized methods for processing and stock tissues aiming to obtain available results for the telepathological interpretation.
Research topics: 1. Cellular signaling pathways in malignant epithelial tumors
No Research area / coordinator Research contracts / budget
(Euro)
1.1
Tumor-microenvironment interactions CS1 Prof. Dr. Carmen Ardeleanu CS3 Biol. Sp. Georgeta Butur CS2.Dr. Dorel Arsene CS3 Dr. Florina Vasilescu CS Dr. Alina Grigore
F20/140.968,04 F70/154586,78
1.2
Intercellular signaling in malignant lymphoma CS1 Prof. Dr. Carmen Ardeleanu CS3 Ass Prof. Dr. Camelia Dobrea Dr. Florina Cionca
F21/ 90.211,59
1.3
Signal transduction anomalies in epithelial malignant tumors CS3 Dr. Cristina Iosif CS3 Dr. Florin Andrei
F40 / 127.272,73
2. Molecular bases of therapy modulation in malignant tumors No Research area / coordinator Research contracts / budget (Euro)
2.1 Breast cancer molecular features Ass.Prof. Dr. Maria Comanescu
CF10 / 68.181,82
2.2 Microsatellite instability of colon carcinoma CS2 Ass.Prof. Camelia Vrabie CS2 Prof. Dr. Maria Sajin
F19 / 159.090,91
2.3 Proteinkinase receptors in lung carcinoma CS3 Dr. Florina Vasilescu
F69 / 45.271,59
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3. Genotypic profiles variability in neoplasia No. Research area / coordinator Research contracts / budget (Euro)
3.1 Molecular genetics in epithelial malignant tumors Senior Res Prof. Dr. Gianni Bussolati Prof. Dr. Carmen Ardeleanu
CF8 / 1.231.125,00 F24/150.000,00
4. Phenotypic and genotypic profiles in inflammatory and degenerative diseases
No Research area / coordinator Research contracts / budget
(Euro)
1.1
Proapoptotic and antiapoptotic factors CS1 Prof. Dr. Carmen Ardeleanu CS3 Biol. Sp. Georgeta Butur CS Dr. Alina Grigore
F22/102.272,73
1.2
Cellular interactions in autoimmune diseases CS1 Prof. Dr. Carmen Ardeleanu CS3 Ass Prof. Dr. Dana Terzea Dr. Florina Cionca
F26/ 93.429,55
1.3
Inflammatory disease of bowel Ass.Prof. dr. Gasbriel Becheanu CS3.Dr. Cristina Iosif
F72/ 45.454, 56 F73/29.021,82 F74/40.590,73
Research team Name Laboratory Name Laboratory CS1 Carmen Ardeleanu Head of pathology
Department Biol. Gaina Gisela
Histopathology Department
CS 3 Biol sp. Georgeta Butur
Head of Diagnostic Center Team coordinator
CSIII. Dana Cristina Terzea
Histopathology Department
CSI Prof. Gianni Bussolati Histopathology Department CSII Dorel Eugen Arsene Histopathology Department C3 Florina Vasilescu Histopathology Department CSII Camelia Vrabie Histopathology Department CS 3 Camelia Dobrea Histopathology Department TS. Florina Alexandru Histopathology Department C3 Cristina Iosif Histopathology Department TS. Alina Anghel Histopathology Department CS Alina Grigore Histopathology Department TS. Valentina Muntean Histopathology Department Dr. Gabriel Becheanu Histopathology Department TS. Elena Ion Histopathology Department Dr. Maria Comanescu Histopathology Department TS. Tatiana Nora Petre Histopathology Department Res Assist Adina Balan Histopathology Department Ts. Florina Manda Histopathology Department Cs. Dr. Georgeta Cardos Histopathology Department TS. Daniel Anghel Histopathology Department CSII. Maria Sajin Histopathology Department TS. Monica Haghighat Histopathology Department Dr. Florina Cionca Histopathology Department ASC Staicu Viorela Histopathology Department
PhD students: molecular biology of nonsmallcell lung carcinoma; breast carcinoma molecular features in young women; genic anomalies in colon carcinomas
Post doc: Advanced studies in molecular biology and gene profiling by gene microarray in breast triple negative breast carcinoma.
Training: 1. Systemic pathology , European School of pathology, Craiova: pancreas and liver– 2007 ( 3 researchers), gastro-intestinal pathology – 2008 (4 researchers), gynaecological pathology – 2009 (3 researchers), male genito-urinary tract pathology – 2010 (3 researchers), thyroid pathology – 2011 (3 researchers); 2. Agilent Gene microarray training for theory and practical applications (3 researchers). 3. Stem cell implication in regenerative medicine, London, 06.10.2011 (1 researcher). Methodological approach o To apply in surgical and molecular medicine the know-how of our research team in human pathology,
cellular and molecular biology
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- Histopathology , cytopathology, histochemistry, immunohistochemistry, in situ hybridization (fluorescent and chromogenic), PCR, Real-time PCR, reverse -transcription PCR, PCR array, gene profile microarray (morphological analysis of tumors, immune phenotyping on archived tissue, for cell differentiation, typing secreting cells, identification of intercellular and intracellular signaling factors, identification of diagnostic, prognostic and predictive factors in tumors, cellular activation and proliferation biomarkers, apoptosis, amplification of genome sequences in infections and tumors, genes mutations, gene profiles of tumors), etc.
Infrastructure Histopathology unit: accreditation according to SR EN ISO 15189 for histopathology and immunohistochemistry (microtome, automatic tissue processor, vacuum automatic tissue processor, scientific microscopes, professional microscopes, routine staining machine); Laboratory of Immunohistochemistry unit: (immunostainers, water baths, microwave oven, automatic cover splipping machine); Hibridization compartment (hybridization plate, fluorescence microscope – Nikon 800, water bath); Molecular diagnosis compartment (termocyclers, real-time PCR automatic system, GEL-Doc, Nanodrop, nucleic acids extractor). International project proposals Proposal full title: Molecular workflow for the effective detection of multiplex HPV markers in cervical cancer patients Proposal acronym: CERVIFLEX, Type of funding scheme: Collaborastive Project Small and medium-scale focused research Work programme topics addressed: HEALTH.2010.1.2-1 Name of the coordinating person: Prof. Dr. Giorgio Stanta Bilateral Italian-Romanian project: Impact of immuno- and geno-typing for improving diagnoses and planning treatment of human tumors. SUPPORT ACTION FOR BILATERAL
COOPERATTION, 2005-2008
TASTE: Telepathological assessment of histopathological and cytological techniques (financed) Education and Culture-Lifelong Learning Programme, 2011-2014, Pr. Nr. 519108-LLP-2011-1-IT-KA3-KA3MP Partner institutions 1. University of Medicine and Pharmacy “Carol Davila”, Bucharest; 2. University of Bucharest; 3. University of Medicine and Pharmacy Craiova, 4. “Gr.T.Popa” University of Medicine and Pharmacy, Iassy, 5 “Victor Babes” University of Medicine and Pharmacy, Timisoara, 6 “Ion Cantacuzino” National Institute of Immunology and Microbiology, 7 Universita degli Studi di Torino, Italy, 8 Roche Pharma Publications 6 ISI publications A1,A2,A14,A28, A26,A71 We were involved in national project regarding cellular signaling pathways in malignant epithelial tumors; molecular basis of therapy modulation in malignant tumors; genotypic profiles variability in neoplasia; phenotypic and genotypic profiles in inflammatory and degenerative diseases Other relevant publications: O2,O6, O10, O11, O16, O31, O40, O53, O61, O65, O95, O33, O43, O54, O62, O66, O109. Development plan I. New research areas: 1. Improvement of molecular signature detection in cancer diagnosis, prognosis and therapy. 2. Applied oncology: Development of molecular approach of archived tumor tissue aiming to offer new potential biomarkers of predictive value in malignant tumors. 3.Development and adapting molecular biology technologies for early detection of malignant tumors and improvement of the therapy. II. Patent submission for new molecular tools in cancer. III. Validation of new experimental models for evaluation of tumor aggressiveness. IV. Introduction of new validated investigation methods in medical practice.
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TEAM 3 - TRANSLATIONAL MEDICAL RESEARCH IN CANCER Team coordinator: CS3. Biol. Sp. Georgeta Butur, Head of Diagnostic Center Mission: translation of high level technology in medical practice to improve survival and life quality of cancer patients Research focus: Development of molecular diagnosis Implementation of new prognostic and predictive biomarkers in malignant tumors Aplication of advanced methods for identification of molecular targets in cancer therapy.
Research topics: 1. Molecular identification of etiologic factors in infections and associated tumors
No Research area / coordinator Research contracts / budget
(Euro)
1.1
Molecular diagnosis of mycobacterial infections CS1 Prof. Dr. Carmen Ardeleanu CS3 Biol. Sp. Georgeta Butur Res. Ass. Biol. Sp. Diana Teletin
F23/22.727,27
1.2
Hepatitis viruses implications in chronic lymphoproliferations CS3 Ass Prof. Dr. Camelia Dobrea CS3 Biol sp. Georgeta Butur
F18/ 39.897,73
1.3 Gene E4 expression of HPV in cervical lesions CS3 Dr. Florin Andrei
F68 / 81.433,86
2. Prognostic and predictive biomarkers in epithelial malignant tumors No Research area / coordinator Research contracts / budget (Euro)
2.1
Biomarkers in malignant tumors CS3 Dr. Florin Andrei CS1 Prof. Dr. Carmen Ardeleanu
F67 / 14.09,91. F24/150.000
2.2
Therapeutic targets in nonsmall cell lung carcinoma, liver disease, melanoma CS3 Dr. Florina Vasilescu CS3 Dr. Camelia Dobrea CS1 Prof. Dr. Carmen Ardeleanu
F70 / 154.586, 78 F17/113.640 F25/45.450
2.3 Prognostic markers in hepatocellular carcinoma, melanoma Asso. Prof. Mariana Costache Asso. Prof Dr. Gabriel Becheanu
F88/121.990 F90/16.540
F71 / 56.818,18
2.4 Biomarkers of renal carcinoma involved in therapy Dr. Mihaela Mihai
F95/ 68.181,82
3. Therapeutic targets in malignant tumors No. Research area / coordinator Research contracts / budget (Euro)
3.1 High-throughput metodologies in personalized oncology) Senior Res Prof. Dr. Gianni Bussolati
CF8 / 1.231.125,00
3.2 Molecular targets in the therapy of malignant tumors CS1 Prof. dr. Carmen Ardeleanu CS3 Biol.sp. Georgeta Butur
Medical services contracts (ROCHE)
3.3
Immunohistochemical biomarkers in malignant tumors therapy CS1 Prof. Dr. Carmen Ardeleanu CS3 Dr. Florina Vasilescu, CS3 Dr. Camelia Dobrea, CS3 Dr. Florin Andrei, CS3 Dr. Cristina Iosif , CS Dr. Simona Enache, CS Dr. Alina Grigore, Ass. Prof. Dr. Maria Comanescu
Medical services contract with Pharma St. Invest., Medical Insurance
Agencies (Bucuresti,Dambovita, Pitesti, Prahova, Vrancea, Buzau, Ilfov, Bacau, Giurgiu, Calarasi)
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Research team Name Laboratory Name Laboratory CS1 Carmen Ardeleanu Head of pathology
Department Histopathology Department
CS 3 Biol sp. Georgeta Butur
Head of Diagnostic Center Team coordinator
Res Ass. Staicu Viorela Histopathology Department
Histopathology Department Biol. Gaina Gisela Histopathology Department C3 Florina Vasilescu Histopathology Department TS. Catalina Culda Histopathology Department CS 3 Florin Andrei Histopathology Department TS. Florina Alexandru Histopathology Department CS 3 Camelia Dobrea Histopathology Department TS. Alina Anghel Histopathology Department C3 Cristina Iosif Histopathology Department TS. Valentina Muntean Histopathology Department CS Simon Enache Histopathology Department TS. Elena Ion Histopathology Department CS Alina Grigore Histopathology Department TS. Tatiana Nora Petre Histopathology Department CS Maria Neagu Histopathology Department TS. Daniel Anghel Histopathology Department Dr. Mihaela Mihai Histopathology Department TS. Monica Haghighat Histopathology Department Dr. Mariana Costache Histopathology Department TS. Elena Naita Histopathology Department Res Assist Adina Balan Histopathology Department Dr. Simona Chirlomez Histopathology Department Res Ass. Diana Teletin Histopathology Department TS. Georgiana Preda Histopathology Department Dr.Valentin Enache Histopathology Department TS. Georgeta Melinte Histopathology Department Histopathology Department TS. Ene Eugenia Histopathology Department PhD students: breast cancer stem cells, molecular features in GISTs, Parasitic infections in humans, phenotypic and genotypic aspects in renal cell carcinoma, Endometrial carcinoma - immunohistochemical and molecular characteristics. Training: ESOT 2009, Paris (2 researchers), International course of pathology of digestive tract, Bucharest, 2008 (5 researchers), 2009 (4 researchers), 2010 (6 researchers), 2011 (3 researchers), Diagnostic histopathology of soft tissue tumors, Treviso Italy, 2008 (4 researchers), Breast pathology, Harvard Medical School, Boston, 2007 (2 researchers). Methodological approach
- To improve the diagnostic in pathology of tumors Histopathology , cytopathology, histochemistry, immunohistochemistry, in situ hybridization (fluorescent and chromogenic), PCR, Real-time PCR, (morphological analysis of tumors, immune phenotyping on archived tissue, for cell differentiation, typing secreting cells, identification of intercellular and intracellular signaling factors, identification of diagnostic, prognostic and predictive factors in tumors, cellular activation and proliferation biomarkers, amplification of genome sequences in infections and tumors).
- To collaborate with medical institutes and clinical hospitals and ambulatories at national and international level, with complementary expertise using high throughput metodologies.
Infrastructure Diagnostic center for translation of know-how and the results of research as services to regional and local health units, aiming to a higher quality of life and to improve the policy of healthcare by personalized therapy . Histopathology unit: accreditation according to SR EN ISO 15189 for histopathology and immunohistochemistry (microtome, automatic tissue processor, vacuum automatic tissue processor, scientific microscopes, professional microscopes, routine staining machine); Laboratory of Immunohistochemistry unit (immunostainers, water baths, microwave oven, automatic cover splipping machine); Hibridization compartment (hybridization plate, fluorescence microscope – Nikon 800, water bath); Molecular diagnosis compartment (termocyclers, real-time PCR automatic system, GEL-Doc, Nanodrop, nucleic acids extractor).
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International project proposals Characterization of early disseminating tumourigenic breast cancer cells with CD44+CD24-/low phenotype and their microenvironment in the bone marrow and bone of patients with Breast cancer Proposal acronym: METASTEM; Type of funding scheme: Collaborative Project: Small or medium scale focused research project Work programme topics addressed: HEALTH-2007-2.4.1-6: Understanding and fighting metastasis, Name of the coordinating person: Prof. Dr. T. Bauernhofer,
You have submitted a proposal to the Electronic Proposal Submission System. Your proposal is now stored on the EPSS system with number 201269 for subsequent evaluation by the Commission. (nonfinanced).
Partner institutions 1. University of Medicine and Pharmacy “Carol Davila”, Bucharest; 2. Medical services contract with Pharma St. Invest.,3 Medical Insurance Agencies (Bucuresti,Dambovita, Pitesti, Prahova, Vrancea, Buzau, Ilfov, Bacau, Giurgiu, Calarasi, 4. “Gr.T.Popa” University of Medicine and Pharmacy, Iassy, 5 “Victor Babes”University of Medicine and Pharmacy, Timisoara, 6“Ion Cantacuzino” National Institute of Immunology and Microbiology, 7. Universita degli Studi di Torino, Italia, 8 Roche Pharma Publications
1. Diagnosis of difficult cases: A1,A2,A26 - we were involved in national project regarding biomarkers in malignant tumors (lung, breast,
malignant lymphomas) and degenerative disease. - Other relevant publication: O34, O44, O57, O63, O81, O35, O36, O48, O59, O82, O37, O39, O135,
O136. Development plan I. New research areas: 1. Development and introduction in the Diagnosis Center of new molecular tools (RT-PCR, microRNA, PCR-array) applied on archived tumor tissue for a more accurate identification of new molecular targets in personalised therapy. 2. Development of multi-skill teams (pathologists, genetists, biochemists, biologists, oncologists, biophisicists, biostatisticians), for molecular diagnosis of malignant tumors by means of high-throughput technologies. II. Validation and patent submission for newly developed molecular tools in the diagnosis and prognosis of cancer. III. Theorical and practical training of pathologists aiming to improve the diagnosis in cancer for applying novel personalized therapies.
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TEAM 4 ‐ ULTRASTRUCTURAL PATHOLOGY LABORATORY The Ultrastructural Pathology laboratory (UPL) is fully equipped laboratory for transmission electron microscopy with modern ancillary equipment and a new FEI Morgagni 100 kV Transmission Electron Microscope (acquired in 2008) for ultrastructural investigation in diagnosis and research. Since 2009, UPL hosts also a Tecnai G2 Spirit BioTWIN Transmission Electron Microscope with single tilt holder which undoubtedly will attract great scientific projects focused on data collection for electron tomography. This technique allows 3D reconstruction of large molecules, organelles and small cells. The UPL is headed by Mihaela Gherghiceanu MD, PhD who has extensive experience in both diagnostic and research applications of electron microscopy and electron tomography. Additional expertise is provided by Eugen Mandache MD, PhD who has over 40 years of experience in the ultrastructural evaluation and diagnosis. He was head of the laboratory until 2010. Technical support is provided by three highly trained electron microscopy technicians and more than 400 samples are processed for ultrastructural investigation for diagnosis or research. UPL TEAM AND ASSOCIATE RESEARCHERS
Mihaela Gherghiceanu, MD, PhD (48 ISI papers; 557 ISI citations, h-index 15) Eugen Mandache, MD, PhD (25 ISI papers; 169 ISI citations, h-index 6) Mihail Hinescu MD, PhD (30 ISI papers; 421 ISI citations, h-index 11) Elena Moldoveanu, PhD (28 ISI papers; 82 ISI citations, h-index 5) PostDoc: Laura Suciu MD, PhD; Marta Daciana PhD PhD students: Gabriela Catalin Biol; Catalin Manole MD
EQUIPMENT - Ultrastructural analysis _ electron microscopy and tomography. Transmission electron microscopes: Morgagni 268 FEI, 100 kV with CCD; Tecnai G2 BioTwin Spirit FEI, 120 kV, single tilt holder with CCD. Ancillary electron microscopy equipment: RMC XL ultra-microtomes; UV/Cry-chamber EMS; Leica cryo-substitution system, diamond knifes, ovens, etc. - Identification and localization of molecules by immunofluorescence: microscopes Nikon E 600 with UV, ZEISS CCD 11Mp; Nikon 200; Leica cryotom. - Image processing: photographic laboratory; 4 Dell and 1 Siemens computers; scanners; software for image analysis (iTEM Olympus, FEI Explore3D, Amira Visage Imaging). DIAGNOSIS The UPL is a nationally recognized, reference center for diagnostic renal pathology and electron microscopy. Diagnostic services are available and include diagnostic evaluation of kidney, muscle, nerve, skin, liver and other
biopsy material (200 specimens/year). In addition to routine ultrastructural techniques, we offer specialized immunofluorescence analysis of biopsy specimens (e.g., kidney, skin). RESEARCH Research projects carried out in the last 5 years by senior researches of UPL were financed with more than 1.000.000 euro. Based on the results obtained from the ultrastructural studies performed in UPL, 36 articles (245 ISI citations) have been published in international journals in the last 5 years. Ultrastructural analysis for “Telocytes” project has been performed in UPL.
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COLLABORATIVE PROJECTS (preliminary studies)
‐ Ultrastructure of hESC and iPSC derived cardiomyocytes ‐ collaborative project with Prof. Ofer Binah
from the Department of Physiology; Ruth and Bruce Rappaport Faculty of Medicine, Technion Israel
Institute of Technology, Haifa, Israel
‐ Zebra fish heart regeneration ‐collaborative project with Christopher Antos, Ph.D. laboratory Center for
Regenerative Therapies; Technische Universitaet Dresden; Germany
‐ Engineered heart tissue ‐ collaborative project with Prof. Changyong Wang from the Tissue Engineering
Research Center, Academy of Military Medical Sciences, Beijing, China
‐ Telocytes in human heart pathology ‐ collaborative project with Prof. Shengshou Hu from the Center for
Cardiovascular Regenerative Medicine, Peking Union Medical College, Chinese Academy of Medical
Sciences, Beijing, China
FUTURE DIRECTIONS Infrastructure investment plan and strategy
Correlative Microscopy at cryo‐temperatures
Integrated or correlative microscopy attempts to combine multidimensional information from complementary
techniques to bridge the various resolution gaps and thus to be able to integrate structural information gathered
from multiple levels of the biological hierarchy into one common framework. Cryo‐fluorescence microscopy for
example can be exploited to navigate the cellular landscapes for features of interest before zooming in on these
areas by cryo‐electron tomography. It offers an independent and unambiguous confirmation of the identity of the
investigated features. For correlative studies we aim to acquire a cryo‐holder and cryo‐ultramicrotome. We also
envisage the acquisition of a state‐of‐art confocal laser scanning microscope with 3‐5 channels for simultaneous
detection of multiple markers in multi‐labelling protocols.
Research focus Cellular and molecular determinants of heart regeneration Stem cell therapy for cardiac diseases has been started before an intrinsic regenerative capacity of heart to be proved and accepted. The dogma that mammalian heart is terminally differentiated organ has been challenged by the reports of few types of resident cardiac stem or progenitor cells. Moreover, a new type of interstitial cell – telocyte - has been described in the adult heart and one important role seems to be nursing stem cells and progenitors in the cardiac stem cell niches. We plan to study the cellular and developmental biology of cardiac stem niches and their involvement in cardiac renewal considering that basic mechanisms governing its physiology are still unknown. By extensive ultrastructural investigation (electron tomography included), confocal microscopy and miRNAs detection we plan a basic research of telocytes and cardiac stem cell niches in normal, ageing and diseased mammalian heart. We also will run a comparative study of regeneration in mammalian and zebrafish (known to have high regenerative capacity) injured hearts. We will try to answer major question: there are one or more types of cardiac stem cell; which cells are mandatory for cardiac renewal; which factors are most important in stem cell differentiation; how newly formed cardiomyocytes are integrated in contractile myocardium; how all these are challenged in diseased heart? All these questions must be answered before an effective cell therapy could be envisaged.
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ULTRASTRUCTURAL PATHOLOGY LABORATORY DEVELOPMENT PLAN I. RESEARCH AREAS
1. Basic research in fundamental mechanisms of cardiac regeneration - from stem cell to heart tissue The identification and classification of somatic stem and progenitor cells that reside in the adult heart. The definition of heterocellular networks that direct stem cells toward a cardiac fate. Comparison of specific cardiac and hematopoietic stem/ progenitor cells by correlative microscopy
(fluorescent and ultrastructural analyses). The establishment of fate-mapping strategies to define the contribution of telocytes and selected
stem/progenitor cell populations to the cardiac lineage during development and after myocardial injury.
2. Structural and molecular characterization of intercellular communication in the interstitial space Spatial structure in intercellular interactions - electron tomography of classical junctions Molecular and structural characterization of ‘atypical’ hetero-cellular junctions in the interstitial
space by correlative microscopy 2. Collaborative projects
Specialized assistance for the research projects involving electron microscopy / tomography (research design and technical expertise): virus ultrastructure and virus -cell interaction; nanoparticles interaction with human cells; induced pluripotent stem cells characterization and tissue integration, etc.
development of ongoing international collaborative projects
II. HUMAN RESOURCES DEVELOPMENT Researcher (junior or senior post-doc) for data collection, computer vision & image processing for
electron tomography of cells and macromolecules Electron Microscopy Research Technician to oversee the laboratory's facilities; to assist and train
visitor researchers
III. INFRASTRUCTURE DEVELOPMENT FOR CORRELATIVE MICROSCOPY confocal laser scanning microscope cryo-holder for TECNAI BioTwin cryo-ultramicrotome
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TEAM 5 PROTEOMIC-BIOMARKERS Team coordinator: CS2 Dr. Cristiana Tanase,
I. The quality of the results of the research activity Research focus: Advanced proteomics: in discovery and application of proteomics biomarkers.
“Core concept”: application of multiple proteomics technologies/platforms in “biomarker driven” research in diagnostics and pharmacology. Research areas:
- biomarker discovery and application as powerful diagnostics instruments in cancer, - proteomics biomarkers in pharmacological research - proteomics in the evaluation of environmental risks for human health, - integration in European Research Area and medical services for biochemistry analyses.
Approach: Multidisciplinary approach, based on integrating proteomics research with other key fields, generating
specific investigation platforms, addressing biomarker research in diagnostics, pharmacology and toxicology. To apply in clinical diagnostics, pharmacotoxicology the know-how of our research team in proteomics, pathology, immunology, cellular and molecular biology, bioinformatics. Key technologies integrated: SELDI-TOF-MS, 2D-DIGE electrophoresis, multiplex-xMAP analysis, protein microarrays, ELISA, cell-cultures. Goals: o To provide and translate research results in the field of biomarkers discovery (mainly in cancer)
into clinical applications; transferring towards hospitals, Ministry of Health and Health Insurance House of technologies/protocols and/or services for the determination of biomarkers.
o To expand and integrate biomarkers research to other disease areas that may benefit from the use of biomarkers in diagnostics, patient stratification, monitoring, etc.
o To collaborate within consortia joining institutions with complementary expertise and multidisciplinary teams to apply individual and/or panels of biomarkers in the fields of molecular diagnostics, pharmacology and risk assessment.
o Preparedness for the integration in European Research Area Expertise: proteomics techniques (mass spectrometry, electrophoresis, multiplex-analysis, immunoassays, cell cultres, (immuno)-toxicology, implementation of techniques for biomarker detection (cytokines, chemokines, growth factors, signal transduction molecules, protein profiling, nucleic acids), bioinformatics. The state-of-the-art technology available in these laboratories and the expertise of the team allow complex, interdisciplinary studies Research projects:
The Proteomics team is involved in 3 international ongoing projects in cancer research and drug testing: CF14 - POS CCE 2.1.2, 152 “Proteomics technologies for cancer biomarkers discovery”; CF11 - bilateral cooperation Romania- China;, “Biomarker discovery in digestive tract cancer and skin melanoma using proteomic approaches” and CF16 - FP7- PIRSES-GA-2008-230816 “Natural Antidiabetic and Antihypertensive Drugs. The team was involved in 19 national projects, as follows: cancer 7, pharmacotoxicology 6, risk assessment 6, networking – 3. Scientifc outcomes:
The results materialized in 15 articles, out of which 10 are published in journals with non-zero relative Article Influence Score (cumulative score: 17,70719), 6 books/book-chapters at international publishers, over 30 presentations at prestigious international conferences with ISI indexed abstract books, of which 7 as invited speakers, 1 certified patent and 2 submitted patents.
Relevant results: (1) The role of Caveolin-1 in cancer progression: we contributed to the annotation of this gene in
cancer pathology (2) Evaluation of specific markers for proliferation, apoptosis and angiogenesis in cancers (3) Key signalling molecules and the main microRNAs in pituitary and, digestive tumours
respectively. (4) Importance of immune markers in the diagnosis, prognosis and therapy monitoring of cutaneous
melanoma. (5) Integration of proteomic biomarkers with in vitro models for the assessment of safety and
efficacy of new potential drugs
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Strategic scientific objectives and directions The major target of the group is to enhance its performance in biomarkers studies, by continuing to develop its output in proteomics biomarkers research. As established priorities for the medium and long term, the team identifies: a) Diagnostic “omics” – development of complex, high performance biomarker based tools with
application in the fields of diagnostics, monitoring, therapy optimization and personalization for cancer, to foster the transfer the approach in other major diseases that will benefit of similar instruments.
- Application of “Omics” technologies for high-throughput biomarker studies - Signal transduction and other regulatory mechanisms in cancer and cancer stem cells - Systems medicine approaches in biomarker discovery (integration of proteomics, miRNomics,
interactomics) - Biomarker panels for early diagnostics, optimized and personalized therapies; integration of
proteomic, miRNA and other biomarkers. - Development of “customized” detection instruments - Translational research: transfer of protocols in clinical units
b) Pharmaco”omics” – profiling induced modulation of expression and activation key regulatory molecules (signal transducers, miRNAs, cytokines) addressing oriented and personalized therapies. Creating a “biomarker driven platform” and validated experimental models for the safety and efficacy assessment in pharmacology and toxicology
II. Quality of human resources The group comprizes 8 senior scientists, 5 young scientists (including 3 Ph.D. students), and 3 lab technicians, with and involvement of 6,05 (scientists) and 2,8 (technicians) full time equivalents. Due to the complementarities of basic and advanced trainings, the group is inter-disciplinary, covering expertizes in medicine, molecular biology, proteomics, bioinformatics, toxicology, immunology. The group has an average age of 37.7 years. Name Title Involvment* Name Role Involvment*Cristiana Tanase MD, PhD, CS2 0.60 Mihail Hinescu MD, PHD,
CS1 0.1
Radu Albulescu Biochemist, PhD, CS1
0.25 Monica Neagu Biochem, PhD, CS2
0.25
Stefan Constantinescu
MD, PhD, Prof.
0.50 Mircea Leabu Chem., Ph.D., CS2
0.30
Ionela Daniela Popescu
Chemist, PhD student, CS
0.70 Carolina Constantin
Biochem., PhD, CS3
0.25
Elena Codrici Biologist, PhD student, CS3
0.80 Irina Radu Technician 0.80
Lucian Albulescu Biochemist, PhD student
0.70 Nicoleta Constantin
Technician 1.00
Simona Mihai MD, RA 1.00 Nicuta Lopazan Technician 1.00 Alina-Ionela Nita Economist,
RA 0.80
*see the structure of Research Teams Continuous education and training: An estimated 6 persons/month spent in the last years for training, covering advanced laboratory techniques in proteomics, bioinformatics etc. Examples of training courses: International course Advanced Proteomics, St.George’s Medical Biomics Centre, University of London, Protein Protein Interaction, European Institute of Bioinformatics, Cambridge, England (2011), SELDI-TOF-Advanced Training Course, Biorad-Paris (2010), ProteinChip SELDI-Basic Training Course, USA (2008), Luminex IS 2.3 Basic Training and Luminex Fundamental Assay Techniques, Oosterhout, The Netherlands, 2D electrophoresis training, Prague, Protein Arrays for Biomarker Discovery and Protein Expression Profiling and Profiling Kinases for Disease Biomarker and Drug Target Discovery, Amsterdam (2007). Bursaries: FEBS 2010 Gothenburg, Sweden (2 persons), FEBS 2011, Turin, Italy (2 persons)
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Exchanges of Personnel: PIRSES-GA-2008-230816 (CF16 Ongoing
Mobilities senior scientists: Invited speakers & Chairmen – Molecular Diagnostics Europe, 2011, London; Cancer Proteomics, 2010, Berlin; Molecular Diagnostics Europe, 2010, Hannover; European biomarkers Congress, 2010, Florence; Proteomics Europe Congress, 2009, Barcelona; BIT Life Sciences’ and Molecular Diagnostics, 2009, Beijing
III. Quality of Infrastructure and degree of exploitation
Based on a coherent policy, the group established and maintained a relevant rate of development of research infrastructure, that parallels the progression of its scientific outcome. Thus, the group established capabilities of research in proteomics that allows multiple, complementary investigations, such as de novo discovery of novel biomarkers (supported by Mass spectrometric, bidimensional electrophoresis and DIGE platforms), multiplex quantitative assays by fluorescence (Luminex xMAP and Luminex xTAG) for cytokines, growth factors, hormones, signal transductors, miRNA, etc). Also, other techniques such as ELISA assays, Western blot, on chip electrophoresis and cell culture assays are available and applied by the group. Domain Equipment Level of exploitation Year of purchase
2D electrophoresis 25% 2007 2D-DIGE (Typhoon 9000) <25% 2011 SELDI-ToF-MS 75% 2008 Western blotting 25-50% 2007 Multiplex xMAP® technology 100% 2007 Protein microarray < 25% 2010 Complete ELISA lines 100% 2007 MiniVIDAS 100% 2007
Proteomics
HITACHI 912 100% 2005 xCELLigence 25% 2010 Complete unit for cell culture 75% 2007-2009 In vitro
assays Microplate Multimode Detector, Anthos Zenyth 3100
75% 2009
Most of the existing equipment is purchased and installed after 2007, and based on functionally proven “state of the art” technologies for both hardware and software components. Validation of the results obtained using the key equipments (thus, of whole “operating chains” for each specific application) is a standard procedure of the group. Interlaboratory comparisons (where such schemes are available) or staff exchange/visits in other laboratories (such as at Biorad labs in Malverne, US, or in “Biomics Center” at St. George’s University, London, UK While mostly used (60% of the functioning time) for the projects coordinated by the senior members of the group, the research infrastructure was also used in projects conducted by other groups, in which individual members of the groups were involved (ca. 30%), while a quota was reserved also for activities involving visiting scientists (e.g. in the NAAN project, during 2010-2011, when 5 scientists visited and worked for 1 month each in the laboratory. International collaboration – Biomics Medical Centre, Saint George’s University of London; Beijing Institute of Genomics, Chinese Academy of Science and BioRad Proteomic Research Center, Malvern, USA and in the FP7 project NAAN – Graz University, University of Lecce, Sekem Egypt.. Recognition at national and international level: The group members are presently involved in international and national activities, as follows: - National Expert in The member State Group of Innovative Medicines Initiative – Joint Undertaking (IMI-JU), Expert in the Program Committee FP7 Health of the EC, Expert evaluators for the FP7 (themes Health - 2010, 2011 calls, NMP - 2011 call), Expert evaluators for the programme “EuroNanoMed”, (2010, 2011 calls), Expert-evaluators for National Plan 2 and for CNCSIS competitions. - Board member of journals: Recent Patents on Biomarkers; Bentham Science Publisher Ltd.), Journal of Immunoassay and Immunochemistry - Peer reviewers for ISI indexed journals: International Journal of Cancer; Future Oncology; Acta Endocrinologica; Recent Patents on Biomarkers; Journal of Immunoassay and Immunochemistry; Medical Principles and Practice Toxicology in Vitro, Materials Science & Engineering B, JMPR Medicinal Plants Research, Roumanian Biotechnology Letters.
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TEAM 6 - IMMUNOMODULATION-IMMUNODIAGNOSIS The “Immunomodulation-Immunodiagnosis” team is composed by 11 graduated members with different specialties, and 4 auxiliaries, as follows: 1. Ursaciuc Cornel - MD, PhD, senior researcher 2, chief of Immunology Department, head of
the team – 80% 2. Neagu Monica - biochemist, PhD, senior researcher 2, chief of Immunobiology Laboratory –
30% 3. Manda Gina - biophysicist, PhD, senior researcher 2, chief of Radiobiology Laboratory –
30% 4. Ciotaru Dan - biologist, senior researcher 3, chief of Immunopathology Laboratory – 50% 5. Constantin Carolina - biochemist, PhD, senior researcher 3 – 30% 6. Surcel Mihaela - chemist, senior researcher 3 – 70% 7. Huica Radu - MD, Drd, researcher – 50% 8. Dobre Maria - biologist, researcher – 60% 9. Munteanu Adriana - biochemist, researcher – 100% 10. Neagoe Ionela - biologist, researcher – 30% 11. Pirvu Ioana - chemist, assistant researcher – 100% 12. Sorca Silvia - nurse – 100% 13. Caralicea Mariana - nurse – 30% 14. Pisica Mariana - technician – 30% 15. Dumitrascu Georgiana - medicine student, technician – 30% Previous activity: The main directions of research activity during last 5 years implied diverse pathology, addressing directly, or participating in the development of topics such as: Immunomodulatory factors of tumor development Immune markers in melanoma Tumor immunogenomics Predictive biomarkers in inflammatory rheumatic diseases Therapeutic potential of environmental factors from salines and caves Innovative immunotherapies in tumors and autoimmune diseases Markers for diagnosis and prognosis of non-viral hepatitis Pannels of markers applicable in personalized medicine Cytokines and immunomodulation
This activity consisted in 5 original projects (developed as project coordinator) and 9 partner projects (developed as partner project responsive) with the above themes, funded by CEEX and PN2 national research programs, as follows: F29-F37, F45-F47, F80, F114, CF5 (see Annex 1). Besides, the team co-operated with other teams in “Victor Babeş” institute (Cellular pathology, Molecular diagnosis technology transfer, Genetics, Drug development & toxicology, Assay development) and other research groups in “Carol Davila” University of Medicine and Pharmacy, as supplier of testing services. The results of scientific activity can be measured, besides the projects applications, in a list of 22 publications, like this: 5 ISI articles, 4 books or book chapters and 13 other relevant publications (see Annex 2). The team is also the contact associated partner in an infrastructure FP7 project:
- FP7 Capacities/2007 – “Biobanking and Biomolecular Resources Research Infrastructure” (BBMRI) – European Commission Grant Agreement Number 212111 - Associate Partner Project responsive Cornel Ursaciuc – 2008-2012
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The team also was/is involved in several scientific service contracts: - Immunofluorescence evaluation of test slides prepared for diagnosis of different serum
autoantibodies cathegories. “Tody Laboratories” SRL Bucharest, 2006-2009 - Experimental model and serum determinations in order to quantify the Simvastatin
effects on systemic inflammation, subclinical heart modifications, and early atherosclerosis in rheumatoid arthritis. “Cantacuzino” Clinical Hospital Bucharest, 2007-2008 - Evaluation of cellular immune status by lymphocyte immunophenotyping and fagoburst test in children with humoral immunodeficiency. IOMC Bucharest, 2007-2011, in progress
- Flow cytometry evaluation of irradiation and flavonoid effects on cell cycle and apoptosis – data aquisition. „Carol Davila” University of medicine and Pharmacy Bucharest, 2010-2011, in progress A constant activity and tradition of this group was performing of humoral and cellular immunodiagnostic tests and hematology tests as currently medical services practised in the “Victor Babeş” Institute's Diagnosis Center. Members of the group also participate in sustaining the Cytometry Unit, Immunogenomics Unit, Biobank and Microbiology Laboratory. At a present, the team is involved as part of the work group in 5 “Victor Babeş” institute’s project proposals and as partner project responsive in other 5 applications by other institutions at the 2011 partner projects call of PN2 national research progamme. Personnel: The group includes various specialists covering the whole area of the biomedical domains (2 medical doctors, 3 biologists, 3 biochemists, 2 chemists, 1 biophysicist). The mean age of the graduated personnel is ~45 years, therefore a partial team rejuvenation is necessary for the next period. We have in view physicians and biochemists, but this is dependent on the financial support of the group during the future time. All the actual staff are good professionals both in laboratory methods and theoretical knowledge and in the same time they are able to perform teaching activity for learning students in immunology and immuno-detection. Part of the staff are involved in formation activities developed through immunology courses organised by Romanian Society of Immunology or human resources projects. The team personnel policy is focused on stimulation of research creativity in order to obtain competitive scientific results. Otherwise the members of the team have attended training courses with the aim of updating their level of scientific information: - Workshop of Fundamental Immunology , Bucharest, Romania, April 2007 - Immune system: genes, receptors and regulation, Hvar, Croatia, 2007 - Molecular biology in diagnosis and epidemiology of infectious diseases. INCDMI ”Cantacuzino” Bucharest, Romania, 2007 - The Course Molecular Diagnostics. The Erasmus Postgraduate School Molecular Medicine, Rotterdam, 2007. - Training in real time PCR at TATAA Biocenter, Prague, 2008 - The Advanced BD FACSCanto™ II and BD FACSDiva™ 6 Training, “Victor Babeş” Institute, Bucharest, Romania, June 15-18 2009 - Epigenetics and new therapies in cancer. ESO-CNIO, Madrid, Spain, May 2007 - Cell culture Seminar, Bucharest, Romania, 2009 - Operator Training on BD FACSCanto II and BD FACSDiva 6.1.2, Heidelberg, Germany, June 2009 - 6th European Course on Clinical Cytometry, Valencia, Spain, September 2010 - 1st EFIS-EJI Intensive Educational Course in Clinical Immunology, Centre de Recherche des Cordelieres, Paris, France, December 1-4 2010. - Autumn Days of Cytometry, Bucharest, Romania, October 2011
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Equipments: The team has performance equipment, and is able to perform a lot of top methodologies in view of accomplish the diverse scientific directions mentioned above. These equipments are located in several units which the team are responsible with: Immunobiology, Immunopathology, Immunoproteomics, Citometry, Immunogenomics, Immunomodulators. A list of purchased equipments are presented in Annex 3. One of the main intension is to upgrade the Immunogenomics unit endowment with a hybridization unit for the gene microarray platform, a new bioanalyser for DNA/RNA evaluation and a supplementary laminar flow hood for nucleic acid extraction. Besides, a refrigerated centrifuge and a small autoclave unit are necessary for Immunopathology. Perspectives: Short- and medium term projects will most likely include acknowledged investigative techniques applied in research or clinical immunological studies: flow cytometry, immunofluorescence, serology, cell culture. Besides, additional investigative techniques such as PCR-array and next-generation sequencing are considered to be developed as an enlargement of immunogenomics branch of laboratory activity. The team will continue several tools of the actual research and also will activate in the diagnosis activity, formation area and biobanking, as: - new scientific projects funded by national programs - co-participants as team work in institute’s proposals - partner institution in consortium projects - service contracts in research or clinical immunology – every kind of immunological investigation performed for beneficiaries from scientific or clinical area. - continued diagnostic work pay activity in institute’s Diagnostic Center - endowment possibilities reached by structural European projects (POS) As constant preoccupations in the research activity will remain: - autoimmune diseases and their diagnosis - tumor immunogenomics - cytokine modulation of cultured cells - serum biomarkers in melanoma and other malignant tumors Other directions will be approached in connection to the results that will be obtained at the partner projects applied to the PN2 national program, whose outcome will appear in spring 2012. Besides, our team will encourage internal and international collaborations (FP projects, bilateral co-operations, service contracts, clinical trials) which implyes the laboratory as a data supplier and scientific consulting collaborator. These will represent a supplementary funds sources for scientific work and researchers mobilities. The above mentioned options will serve also as data sources used for meeting presentations, scientific articles or books.
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TEAM 7 - GENOMICS AND GENETIC DIAGNOSIS
Research interests genetics of neuropsychiatric disorders associated with intellectual disabilities (ID). Multiple genetic anomalies are associated with ID phenotypes, and new data are reported at a high rate. However, the underlying genetic mechanisms are seldom clear. Currently, we focus on the identification of genetic aberrations associated with ID at genomic and cytogenetic level with the aim to further the knowledge regarding the etiopathogeny of complex neuropsychiatric disorders. characterization of genetic and epigenetic alterations underlying the initiation and progression of cancer (hematologic neoplasms and solid tumors); cancer immunogenomics. The genetic abnormalities associated with cancer bear a well-known clinical significance and are of outmost interest in the diagnosis, risk assessment, and management of the disease; therefore, their characterization and understanding brings multiple benefits for the medical science, and ultimately for the patients. Epigenetic studies, due to the reversible nature of epigenetic modifications, have a great potential not only for biomarkers discovery relevant for cancer detection and prognosis, but most importantly for identifying new targets for epigenetic drugs. Presently, we focus on the detection and characterization of these abnormalities at gene, chromosome and genome level, aiming to identify new aberrations and their contribution to the malignant phenotype. We also started to investigate the epigenetic changes in breast cancer cell lines, aiming to extend these studies to breast cancer diagnosis and prognosis.
Our experimental approaches cover molecular cytogenetics, molecular genetics, cell and molecular biology, and genomics (immunogenomics included). The team uses state-of–the art infrastructure for several applications, including: optical microscopy (transmitted light and epifluorescence), molecular cytogenetic techniques (e.g. FISH, mBAND), „in house” preparation of FISH probes using bacterial artificial chromosomes, PCR based techniques, and high-resolution genome wide approaches (DNA microarray). The research strategy is based on using unique patient material obtained through close collaboration with Clinical Departments from major tertiary hospitals. Additionally, our team makes constant efforts to preserve the valuable biological material that it is not used in ongoing studies, for future in depth or large scale approaches.
Research grants Since 2007, the team participated in 6 national projects and 2 bilateral cooperation projects (Romania-France). The projects were granted within national peer-reviewed competitions. No. Research area Research grant ID
no/ funding(Euro) 1 Genetics research of neuropsychiatric disorders
F1, F10/ 386,237
2 Hematologic neoplasms genetic characterization F6, F96/ 213,635 3 Human foetal hepatic stem cells characterization F3/68,181 4 Infrastructure project for genetic research F4/425,452 5 Bilateral Cooperation projects – knowledge and expertise sharing in the
field of genetic/genomic defects of complex pediatric neuropsychiatric disorders
F2, CF1/ Funding for bilateral visits
Infrastructure Up-grading the research infrastructure with state-of-the-art technologies has been the mainstay of our team strategy. A major step forward was made with a grant dedicated to enhancing research capabilities (2007-2009). Consequently, microarray (Agilent platform) and molecular genetics facilities were developed; the light microscopy facility was greatly improved by the acquisition of a motorized optical microscope. Presently, the team uses the following research equipments/facilities: Microarray facility: Agilent DNA Microarray C Scanner with Surescan, Agilent Scan Control, Feature extraction and Genomic Workbench Software; Agilent BioAnalyzer 2100; Molecular genetics facility: Corbett Real Time PCR, Corbett Palm Cycler PCR; Nucleic acid isolation and manipulation facility: chemical hoods, water baths, cooling centrifuges,spectrophotometer, BioDoc transilluminator, freezers, ultrafreezers; Cell culture facility: safety cabinets, incubators; Light microscopy facility: motorized Axio Z1 Zeiss microscope with examination in transmitted light bright field and epifluorescence, equipped with high resolution monochrome cooled CCD camera; Metafer and
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Ikaros software for scanning, automatic detection of metaphases, capture, karyotyping, FISH (standard, M-FISH, m-BAND, Q- FISH, CGH).
Research team Name Laboratory Aurora Arghir, CS3 Medical Genetics Laboratory Andreea Tutulan-Cunita, CS 1)** Medical Genetics Laboratory Sorina Papuc, CS* Medical Genetics Laboratory Magdalena Budisteanu, MD Medical Genetics Laboratory Cornel Ursaciuc, CS2 Immunopathology Laboratory Monica Dobre, CS Immunopathology Laboratory Radu Huica, CS3 * Immunopathology Laboratory Sevinci Pop, CS3 2)** Cell Biology Laboratory Valeriu Cismasiu, CS 3)** Cell Biology Laboratory Georgeta Cardos, CS 4)** Pathology Department Gisela Gaina CS3 Pathology Department Maria Neagu*, CS Pathology Department Angela Petrescu, CS 3 IT Department Agripina Lungeanu, CS1*** Medical Genetics Laboratory Ioana Borcan, Technician Medical Genetics Laboratory Marioara Cristea, Technician Medical Genetics Laboratory * PhD students **1) Specialization in molecular biology and genetics (Master degree 2000-2002/ PhD 2002-2005 - Hiroshima University, Japan; Postoctoral fellowship 2005-2006 - Manchester University, UK); 2) Doctoral fellowship: 2000-2002, Postoctoral fellowship: 2003-2008 University of Illinois at Urbana-Champaign, USA, Department of Cell and Developmental Biology; 3) Specialization in molecular biology, protein biochemistry and cell biology within several postdoctoral fellowships (Albany Medical Center, NY, USA; Stem Cell Center, Lund, Sweden; Wetherall Institute of Molecular Medicine, Oxford, UK); 4) Doctoral fellowship: 2004-2008, Hamburg University, Germany.
*** Retired - 2010.04 Research / clinical collaboration The research activities, focused on detecting clinically relevant genetic lesions in neuropsychiatric disorders and cancer, add data to the body of knowledge and ultimately contribute to the improvement of clinical management of the patients. This is achieved through close collaboration with groups from clinical hospitals such as: “Prof. Dr Alexandru Obregia” Clinical Hospital of Psychiatry, Coltea Clinical Hospital (Hematology Department), Emergency University Hospital Bucharest (Hematology Department), other oncological departments. In order to address the technological challenge of analyzing large amount of data generated by genome-wide high through-put technologies (microarrays) our team recently established collaboration with a bioinformatics/biostatistics group from “Al. I Cuza” University, Faculty of Computer Sciences. Several international collaboration were established by our team with research groups from France (Professor Jean-Michel Dupont team, Cochin Hospital, Paris), Germany (Professor Evelin Schrock team, Carl Gustav Carus Faculty of Medicine, Dresden) and United States (Professor Kenneth Kosik team, University of California, Santa Barbara). A long term collaboration has been developed with Cochin Hospital through two bilateral cooperation (Romania-France) projects focused on genetic defects underlying intellectual disabilities and autistic spectrum disorders, respectively. This collaboration materialized in mutual visits and short-term staff trainings, exchange of technologies, and joint publications. The cooperation with professor Kosik team focus on understanding the pathogenetic mechanisms of neuropsychiatric disorders, such as microdeletion syndromes; it involves the generation of induced pluripotent stem cells (obtained in professor Kosik lab from fibroblasts) with subsequent differentiation into neuronal tissue in order to obtain a more accurate disease model. A recent collaboration was established with Yolanda de Diego team from Carlos Haya Hospital, Malaga, Spain on the topic of fragile X etiopathogeny and the perspective of antioxidative therapeutic strategy. Publications: A49, A72, A93, A94, A95. Training: 1. Advanced training in aCGH (Institute of Clinical Genetics, Carl Gustav Carus Faculty of Medicine, Dresden, Germany) – 1 researcher; 2. Oligonucleotide Microarray training (Institute G. Roussy, Paris, France) – 3 researchers; 3. Clinical Cytogenetics Course (European Cytogeneticists Association, South Tyrol, Italy) - 1 researcher; 4. Courses of Dysmorphology 2006, 2008, 2009, 2011
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(European Society of Human Genetics, Rome, Italy) – 1 researcher; 5. European Course on Genetics of Mental Retardation (European Society of Human Genetics, Braga, Portugal) - 1 researcher; 6. Workshops on laboratory accreditation, internal quality control and external quality assessment, (EuroGentest, Nice, France and Berlin, Germany) – 2 researchers; 7. Project Management Course (AFPA, Bucharest, Romania) – 2 researchers. International project proposals: FP7 – ERC Starting grants, 2010 Call “Integrative analysis of DNA methylation, miRNA and gene expression profiles in idiopathic autism” (not financed); FP7 – ERC Starting grants, 2011 Call “Genome-wide analysis of drug responsiveness in attention deficit hyperactive disorder” (under evaluation); FP7 - HEALTH-2012- INNOVATION-1 „A phase III randomized double blind multicenter clinical trial to investigate the efficacy of the combination of ascorbic acid and tocopherol versus placebo for the treatment of cognitive deficit and behavioral problems in the fragile X syndrome. Functional relevance of the RAC1-GTPase as molecular target” (under evaluation). Perspectives - Since 2007, our team advanced from molecular cytogenetic and molecular genetic studies of individual genes/ chromosomal regions towards genome-wide molecular strategies. The envisaged strategy of our team consist of identification and characterization of constitutional and acquired genomic abnormalities, by genome-wide molecular approaches and use of the resources generated by the Human Genome Project.
Understanding the biological bases of complex neuropsychiatric disorders is one of the most important medical challenges of the present time and most probably of the next years. Expanding usage of whole-genome molecular strategies has given a new quality to the analysis of these disorders. Thus, array-CGH can detect discrete copy-number changes and allows the definition of new clinical syndromes. Additionally, the possibility to combine CNVs data with genotyping information (SNP arrays) provides the advantage of adding high resolution and investigation of copy-neutral events. Our team is involved in searching for clinically relevant genes/genomic regions through array-CGH studies, with further interest in extending our investigations at transcriptomic, epigenetic/epigenomic as well as model organism level. The collaboration with professor Kosik lab will allow us to participate in the development of more accurate disease models for complex neuropsychiatric disorders such as microdeletion syndromes. Cancer is caused by genetic and epigenetic anomalies that alter the balance among cell proliferation, survival, and differentiation. However, only a fraction of the cancer-associated genetic aberrations have been identified. In this context, our team aims at identifying novel oncogenic lesions relevant for pathogenesis as well as studying the cooperation between genetic events. On short and medium term, our team intends to expand the area of interest toward the investigation of acquired uniparental disomies, alterations of gene expression and epigenetic changes in relation with disease onset and progression in cancer (hematologic malignancies, breast cancer, other solid tumors). Identifying and characterizing immunogenomic markers and assessing their diagnostic and prognostic impact is yet another objective of our team. A long term goal is to bring new evidence on the role of microenvironment in tumorigenesis, with special emphasis on mesenchimal stem cells. By analyzing the genetic/genomic abnormalities of tumor-associated cell populations, new factors at interplay in the oncogenic process can be identified and new potential treatment targets be found. While array-CGH is already in place and functioning in our institute, the expansion towards the genome-wide investigation of copy-neutral alterations (e.g. uniparental disomy, loss of heterozygosity) demands an upgrade of the existing technology (Agilent High Resolution Scanner Upgrade License - License for C Scanner upgrade – allows CGH+SNP array as well as scanning of high definition platforms). For adding higher resolution – up to nucleotide level - to the genome-wide investigations, our team foresees developing partnerships with neighbouring institutions (e.g. Carol Davila University of Medicine and Pharmacy and Institute of Cellular Biology and Pathology “N. Simionescu”), as well as foreign institution. By joining expertise and sharing resources (next-generation sequencing technologies available in the above mentioned institutions) our team intend to expand its whole genome molecular strategies towards hypothesis independent mutation screening in cancer and complex neuropsychiatric disorders. In order to exploit both the research and diagnostic potential of array-CGH, its introduction in clinical laboratory as a diagnostic tool is one of our goals. In close collaboration with a bioinformatics team as well as with clinical groups, we intend to device a reliable and sensitive method for the investigation of genomic structural variation in several human pathologies.
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TEAM 8 - NEUROSCIENCES Evolution of the human resources Neuroscience Team composition between 2007 and 2011
Period Name Team position 2007-2011 Bogdan O. Popescu, MD, PhD, senior researcher, neurologist Head of laboratory 2008-2011 Emilia Manole, PhD, senior researcher, biologist Member 2007-2011 Paula Gratiela Chelu, MD Member 2007-2008 Raluca Colesniuc, researcher, biologist Member 2007-2011 Catalin Manole, MD, research assistant, PhD student Member 2007-2011 Dragos Cretoiu, MD, research assistant, PhD student Member 2009-2011 Mihnea Nicolescu, MD, research assistant, PhD student Member 2008-2010 Oana Romanitan, MD, PhD Member 2011 Laura Suciu, MD, PhD Member 2007-2011 Mariana Nicolae, technician Member In 2007-2009, two students were carried out their research work for the undergraduate thesis: Radu Stoica and Maria Tuineag (on effects of antiepileptic drugs on neuronal apoptosis and neuroplasticity). In 2009-2011, Ana Maria Enciu, MD, PhD student, worked in the laboratory for her PhD thesis (Molecular mechanisms in neurodegeneration). Our team we have a balanced distribution of age (mean age of 38, 6 years) and sex (6 women, 4 men). Members of the team have different complementary expertise (both medicine and biology). Dr. Bogdan O. Popescu has graduated a PhD in Neuroscience in Karolinska Institute, Stockholm and works as well as a senior neurologist. The dynamic of the research subjects and directions Models and techniques currently used in laboratory are: cell cultures, animal models of neurodegeneration, light microscopy (phase contrast, fluorescence, confocal), histology, histochemistry, immunohistochemistry, Western blot, ELISA, PCR. The main research directions of the laboratory during this period were: studies of the trophic factor receptors expression in the central and peripheral nervous system, the distribution and expression of the tight junction proteins in the brain, new neurodegeneration models relevant to Alzheimer disease and Parkinson disease. Since 2008, we performed as well studies of distribution and expression of different proteins involved in skeletal muscle and peripheral nerve pathology. The most important achievements Grants For the period 2007-2010 we obtained two research grants (PN II - Partnerships, 2007):
1. PN II 41-013 /2007: Expression and function of tight junction proteins – a study in experimental models and in patients with dementia. Project Director (National Institute of Pathology “Victor Babes”): Bogdan O. Popescu, MD, PhD; Partners: International Center of Biodynamics, Bucharest and Bucharest Emergency University Hospital.
2. PN II 61-019/2007: Implementation and optimization of the technological process of obtaining active therapeutic serum F (ab ') 2 against highly bacterial and viral pathogenic agents. Project Director (National Institute for Microbiology and Immunology “Dr. I. Cantacuzino”): Nadia Bucurenci, PhD; Project Director of the Partner 1 (National Institute of Pathology “Victor Babes”): Bogdan O. Popescu, MD, PhD; Partner 2: University of Medicine and Pharmacy “Carol Davila”, Bucharest.
For the period 2008-2011 we obtained two research grants (PN II - Partnerships, 2008): 1. PN II 42-124/2008: Molecular analysis of the proteins implicated in the main types of
peripheral neuropathies with a demyelinating component. Project Director (National
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Institute of Pathology “Victor Babes”): Bogdan O. Popescu, MD, PhD; Partners: University of Bucharest and Bucharest Emergency University Hospital.
2. PN II 42-133/2008: Cellular and molecular bases of muscle ageing. Project Director (University of Bucharest): Emilia Manole, PhD; Project Director of the Partner 1 (National Institute of Pathology “Victor Babes”): Bogdan O. Popescu, MD, PhD; Partner 2: Colentina University Hospital.
Education Starting with 2011, we contribute to the TDM project (Education of medical personnel - new technologies for health system/molecular diagnosis-POSDRU/81/3.2/S/58819, coordinated by our Institute. The Western blot expert from the Proteomics Section is Dr. Emilia Manole and the Direct Immunofluorescence expert from the Molecular Imaging Section is Dr. Laura Suciu, both from our laboratory. Dr. Bogdan O. Popescu authored one textbook for medical students and two book chapters for clinical neuroscience specialists. He is the Executive Editor of Romanian Journal of Neurology (CNCSIS B+) and Secretary General of the Romanian Society of Neurology as well. Dr. Bogdan O. Popescu served during this period as reviewer for several ISI journals and international grant evaluations (European Science Foundation and American Alzheimer Association). Publications Since 2007, the members of our laboratory published 20 ISI full-text articles and 8 full-text articles indexed in other international data bases and contributed with more than 50 lectures and posters in international and national scientific meetings. National scientific awards Dr. Bogdan O. Popescu - ‘Victor Babeş’ award of the Romanian Academy for medical
research activity – 2007. Dr. Bogdan O. Popescu - ‘Science and Art National Foundation Award of Excellence for
research in the field of Neuroscience and Neuropathology – 2010.
List of ISI publications (2007-2011)
1. Negreanu L, Popescu BO, Babiuc RD, Ene A, Bajenaru OA, Smarandache GC., Duodopa infusion treatment: a point of view from the gastroenterologist., J Gastrointestin Liver Dis. 2011 Sep; 20 (3): 325-7.
2. Enciu AM, Constantinescu SN, Popescu LM, Mureşanu DF, Popescu BO., Neurobiology of vascular dementia., J Aging Res. 2011; 2011: 401604. Epub 2011 Aug 17.
3. Enciu AM, Nicolescu MI, Manole CG, Mureşanu DF, Popescu LM, Popescu BO, Neuroregeneration in neurodegenerative disorders, BMC Neurol. 2011 Jun 23; 11:75.
4. Enciu AM, Popescu BO, Gheorghisan-Galateanu A., MicroRNAs in brain development and degeneration, Mol Biol Rep. 2011 Jun 5. [Epub ahead of print]
5. Popescu LM, Manole E, Serboiu CS, Manole CG, Suciu LC, Gherghiceanu M, Popescu BO, Identification of telocytes in skeletal muscle interstitium: implication for muscle regeneration., J Cell Mol Med. 2011 Jun; 15(6): 1379-92.
6. Popescu LM, Gherghiceanu M, Suciu LC, Manole CG, Hinescu ME., Telocytes and putative stem cells in the lungs: electron microscopy, electron tomography and laser scanning microscopy, Cell Tissue Res. 2011 Sep; 345(3): 391-403.
7. Hinescu ME, Gherghiceanu M, Suciu L, Popescu LM., Telocytes in pleura: two- and three-dimensional imaging by transmission electron microscopy, Cell Tissue Res. 2011 Feb; 343(2): 389-97.
8. Nicolae L, Iacob G, Poparda M, Popescu BO, Case report. Gelastic seizures in a patient with right gyrus cinguli astrocytoma, J Med Life. 2010 Oct-Dec; 3(4): 433-6.
9. Negreanu LM, Popescu BO, Babiuc RD, Ene A, Andronescu D, Băjenaru OA., Cutting the Gordian knot: the blockage of the jejunal tube, a rare complication of Duodopa infusion treatment, J Med Life. 2010 Apr-Jun; 3(2): 191-2.Erratum in: J Med Life. 2011 Jan-Mar; 4(1): 7 p following 123.
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10. Bajenaru O, Tiu C, Moessler H, Antochi F, Muresanu D, Popescu BO, Novak P., Efficacy and safety of Cerebrolysin in patients with hemorrhagic stroke, J Med Life. 2010 Apr-Jun; 3(2): 137-43.
11. Dumitriu A, Popescu BO., Placebo effects in neurological diseases, J Med Life. 2010 Apr-Jun; 3(2): 114-21. Review.
12. Jianu DC, Muresanu DF, Bajenaru O, Popescu BO, Deme SM, Moessler H, Meinzingen SZ, Petrica L., Cerebrolysin adjuvant treatment in Broca's aphasics following first acute ischemic stroke of the left middle cerebral artery., J Med Life. 2010 Jul-Sep; 3(3): 297-307. Erratum in: J Med Life. 2011 Jan-Mar; 4(1): 7 p following 123.
13. Muresanu DF, Alvarez XA, Moessler H, Novak PH, Stan A, Buzoianu A, Bajenaru O, Popescu BO., Persistence of the effects of Cerebrolysin on cognition and qEEG slowing in vascular dementia patients: results of a 3-month extension study, J Neurol Sci. 2010 Dec 15; 299(1-2): 179-83.
14. Hort J, O'Brien JT, Gainotti G, Pirttila T, Popescu BO, Rektorova I, Sorbi S, Scheltens P; EFNS Scientist Panel on Dementia, EFNS guidelines for the diagnosis and management of Alzheimer's disease, Eur J Neurol. 2010 Oct; 17(10): 1236-48.
15. Negreanu L, Popescu BO., Evaluation of successful treatment in achalasia with timed barium esophagogram: revisiting an old friend, J Med Life. 2010 Jan-Mar; 3(1): 64-6.
16. Ghitoiu A, Rusu EC, Slăvoacă D, Aigyul E, Popescu BO., A hypertensive patient with multiple intracerebral hemorrhages due to brain metastases., J Med Life. 2009 Oct-Dec; 2(4): 437-9.
17. Romanitan MO, Popescu BO, Spulber S, Băjenaru O, Popescu LM, Winblad B, Bogdanovic N., Altered expression of claudin family proteins in Alzheimer's disease and vascular dementia brains, J Cell Mol Med. 2010 May;14(5):1088-100.
18. Suciu L, Nicolescu MI, Popescu LM., Cardiac telocytes: serial dynamic images in cell culture, J Cell Mol Med. 2010 Nov; 14(11): 2687-92
19. Suciu L, Popescu LM, Gherghiceanu M, Regalia T, Nicolescu MI, Hinescu ME, Faussone-Pellegrini MS., Telocytes in human term placenta: morphology and phenotype, Cells Tissues Organs. 2010; 192(5): 325-39.
20. Popescu BO, Toescu EC, Popescu LM, Bajenaru O, Muresanu DF, Schultzberg M, Bogdanovic N., Blood-brain barrier alterations in ageing and dementia, J Neurol Sci. 2009 Aug 15; 283(1-2): 99-106. Review.
21. Cretoiu SM, Cretoiu D, Suciu L, Popescu LM., Interstitial Cajal-like cells of human Fallopian tube express estrogen and progesterone receptors., J Mol Histol. 2009 Oct; 40(5-6): 387-94.
22. Suciu L, Popescu LM, Regalia T, Ardelean A, Manole CG., Epicardium: interstitial Cajal-like cells (ICLC) highlighted by immunofluorescence, J Cell Mol Med. 2009 Apr; 13(4): 771-7.
23. Muresanu DF, Alvarez XA, Moessler H, Buia M, Stan A, Pintea D, Moldovan F, Popescu BO., A pilot study to evaluate the effects of Cerebrolysin on cognition and qEEG in vascular dementia: cognitive improvement correlates with qEEG acceleration, J Neurol Sci. 2008 Apr 15; 267(1-2): 112-9
24. Popescu BO., Still debating a cause and diagnostic criteria for Alzheimer's disease, J Cell Mol Med. 2007 Nov-Dec; 11(6): 1225-6
25. Oprica M, Hjorth E, Spulber S, Popescu BO, Ankarcrona M, Winblad B, Schultzberg M., Studies on brain volume, Alzheimer-related proteins and cytokines in mice with chronic overexpression of IL-1 receptor antagonist., J Cell Mol Med. 2007 Jul-Aug; 11(4): 810-25.
26. Romanitan MO, Popescu BO, Winblad B, Bajenaru OA, Bogdanovic N., Occludin is overexpressed in Alzheimer's disease and vascular dementia., J Cell Mol Med. 2007 May-Jun; 11(3): 569-79.
27. Cowburn RF, Popescu BO, Ankarcrona M, Dehvari N, Cedazo-Minguez A., Presenilin-mediated signal transduction., Physiol Behav. 2007 Sep 10; 92(1-2): 93-7 Review.
28. Suciu L, Popescu LM, Gherghiceanu M., Human placenta: de visu demonstration of interstitial Cajal-like cells, J Cell Mol Med. 2007 May-Jun; 11(3): 590-7.
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TEAM 9 - DRUG DEVELOPMENT AND TOXICOLOGY Team coordinator: CS2 Dr. Gina Manda, Head of Radiobiology Laboratory Mission: applied biomedical research in drug development, medicinal chemistry and (immuno)toxicology Research focus: Identification of pathology-relevant drug targets and action mechanism medicinal chemistry: screening & hits to leads (biological activity assessment)
o development and implementation of in vitro assays with particular end-points, for selecting candidate drugs, for defining their action mechanism and safety profile
o assistance of partner research teams (organic and analytic chemists, pharmacists) in designing and producing new compounds (libraries of synthetic or natural compounds), potentially efficient in cancer, autoimmune, cardiovascular diseases etc.
o preclinical studies for these new compounds, both in vitro and in vivo in animal models (selection of candidate compounds by in vitro studies, in vivo proof-of-action studies using relevant animal models, in vitro and in vivo mechanistic studies and toxicological screening)
immunotoxicology o implementation of advanced screening methods for characterizing the biological impact of
xenobiotics on human health (medicines, drugs of abuse, heavy metals, mycotoxins, ionizing radiation etc)
Research topics: 1. Pathologic mechanisms and drug targets
No Research area / coordinator Research contracts /
budget (Euro)
1.1 Biomarkers and molecular targets in cardiovascular diseases Prof. Dr. Elena MOLDOVEANU and CS3 Dr. Daciana MARTA
F49, F50, F51 / 336.381
1.2 Immune targets in the treatment of rheumatoid arthritis CS2 Dr. Gina MANDA
F77 / 73.070
1.3 Integrin – extracellular matrix interactions in cell motility and metastasis CS2 Dr. Mircea LEABU
F108 / 100.676
2. Drug development
No Research area / coordinator Research contracts /
budget (Euro)
2.1 Novel nucleoside analogs for cancer therapy - CS2 Dr. Gina MANDA F81, F82 / 188.653
2.2 Complexes of physiological, divalent transitional metals for cancer therapy - CS2 Dr. Mircea LEABU
F106 / 92.053
2.3 Functionalized nutrients - CS2 Cristiana TANASE F41 /125.000
2.4 Alternative medicines: bioactive phytochemicals CS2 Dr. Cristiana TANASE, CS2 Dr. Mircea LEABU
F38, F52, F53, F105 / 347.780
3. (Immuno)Toxicology
No Research area / coordinator Research contracts /
budget (Euro)
3.1 Immunotoxicology of mycotoxins: impact on the food chain and development of counteracting agents (study in porcine model) CS2 Dr. Gina MANDA
F76, F79 / 48.646
3.2 Heavy metals profiling in biological samples and food CS3 Vasile PREOTEASA
Nucleu Programme
3.3 Radiation-induced genotoxicity CS3 Vasile PREOTEASA
Contract with Cernavoda nuclear power plant
3.4 Geno- and hepatotoxicity of xenobiotics CS3 Bogdan Marinescu
Contract with S.C. ALCEDO S.R.L.
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Publications 2. Pathologic mechanisms and drug targets: 7 ISI publications A13, A17, A20, A24, A36, A83,
A96, O15, 096, 098, 099 (cumulated rAIS 12,32 citations 18). - biomarkers in vascular inflammatory pathology (pulmonary hypertension, heart failure and
antiphospholipid syndrome). In heart failure we add contribution to the recognition of LpPLA2 as a marker of oxidative stress and vascular inflammation.
- in rheumatoid arthritis we highlighted particular networks of adaptive and innate immunity (B lymphocytes-NK cells) and a key role of peripheral monocytes in mirroring disease outcome; we showed that low doses of immunosuppressive agents (leflunomide) may exert a pro-inflammatory action on pathologic monocytes.
3. Drug development: 12 ISI publications A12, A31, A41, A43, A57, A65, A67, A84, A88, A89, A90, O28 (cumulated rAIS 5.69, citatons 12), 1 book chapter (O28).
22 new nucleoside analogs from 3 structural classes were investigated in vitro and 3 lead compounds with anti-cancer activity were identified. A preliminary in vivo study on laboratory animals proved their activity and showed a convenient toxicological profile for 2 of the lead compounds. Compounds will be further developed, including by preliminary results-guided structural changes. We will investigate their therapeutic application as radiosensitizers in cancer therapy and we will submit at least 1 national patent.
4. Toxicology: 3 ISI publications A71, A82, A92 (cumulated rAIS 2.43), 1 book chapter (O19), 071. Accreditation of immunotoxicology assays according to SR EN ISO 15189 (Biochemistry Laboratory) - F56/181.512 Euro Research team Name Laboratory Name Laboratory
CS2 Gina Manda 1)** Team coordinator, Head of Radiobiology Laboratory. Scientific secretary
CS3 Ciotaru Dan Head of Immunopathology Laboratory
CS Ionela Victoria Neagoe Radiobiology Laboratory CS3 Mihaela Surcel Immunopathology Laboratory CS3 Vasile Preoteasa Head of Nuclear Unit CS3 Radu Huica* Immunopathology Laboratory
CS2 Mircea Leabu 2)** Head of Cell Biology Laboratory
CS2 Cristiana Tanase Head of Biochemistry Laboratory
CS3 Sevinci Pop 3)** Cell Biology Laboratory CS1 Radu Albulescu Biochemistry Laboratory CS Andreea Oana Urs* Cell Biology Laboratory CS Daniela Popescu* Biochemistry Laboratory Cristina Mariana Niculite* Cell Biology Laboratory AS Lucian Albulescu* Biochemistry Laboratory Mihaela Andreea Mocanu Cell Biology Laboratory Catalin Filipescu Cell Biology Laboratory CS Alina Grigore Pathology Department
CS3 Daciana Marta Ultrastructural Pathology Laboratory
CS Bogdan Marinescu Head of Animal Care Unit, Laboratory of experimental models
CS3 Gabriela Catalin* Ultrastructural Pathology Laboratory
CS Gheorghita Isvoranu* Animal Care Unit, Laboratory of experimental models
Technicians: Daniela Geogia, Sanda Sima, Cristina Vlad, Maria Paraschiv * 7 PhD students ** 1) Specialization in rheumatoid arthritis (idiotype/anti-idiotype network), INSERM, Paris, France; 2) Post-doctoral fellow and visiting researcher, 2000-2005, University of Western Ontario, London, ON, Canada; 3)Doctoral fellowship: 2000-2002, Postoctoral fellowship: 2003-2008 University of Illinois at Urbana-Champaign, USA, Department of Cell and Developmental Biology. Methodological approach o To apply in medicinal chemistry and toxicology the know-how of our research team in human
pathology, immunology, cellular and molecular biology - cell cultures, flow cytometry (immune phenotyping, cellular activation and proliferation, generation
of reactive oxygen species, apoptosis), protein multiplexing (soluble factors profile, signal transduction pathways), fluorescence and confocal microscopy, transmission electron microscopy, impedance measurements for cell adhesion, radio-assays (biodistribution, cell functions), heavy metals profiling in biological samples by ICP-MS etc.
o To collaborate within consortia joining institutions with complementary expertise and multidisciplinary teams (biologists, biochemists, medical doctors, chemists, pharmacists, biophysicists etc) for developing innovative compounds designed and produced by Romanian R&D teams and for implementing new investigation methodologies for Drug development and Immunotoxicology.
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Available infrastructure Cell culture facilities - class II flow hoods, CO2 incubators, deep freezing unit; Cytometry unit - 2 flow cytometers, fluorescence microscopes, confocal microscope; VIDEOCELL unit - xCELLigence system, BioStation IM, fluorescence microscope, electrophoresis and western blot equipment, gas chromatograph; Proteomics unit (Luminex platform); Histology unit; Animal Care Unit: 2008-2011 accreditation according to SR EN ISO 17025 for B12 and B39 assays; Nuclear unit (certified by CNCAN): Canberra Packard beta-counter, Sorcerer imager; ICP-MS unit: ICP-MS ULTRAMASS 700. Infrastructure upgrading in the Animal Care Unit and in the Cellular Biology Laboratory (877.466 Euro from INFRAS and Capacities Programmes) Collaborations National collaborations: 1. University of Medicine and Pharmacy “Carol Davila”, Bucharest; 2. University of Bucharest; 3. R&D Institute for Chemical-Pharmaceutical Research, Bucharest; 3. Institute for Nuclear Physics and Engineering “Horia Hulubei”, Magurele; 4. Institute of Biology and Animal Nutrition, Balotesti (IBNA); Private companies: Mecro Systems and SC Biotehnos SA; International collaborations: 1. Universite Libre de Bruxelles, Laboratory of Pharmaceutical Chemistry, Brussels, Belgium; 2. University of Thessaly, School of Health Sciences, Biochemistry Department, Greece. International project proposals 1. FP7-NMP-2009-SMALL-3: Nanotechnologies for medical implants (not financed); 2. IMI_Call_2009_5: Aberrant Immunity in Chronic Immune-mediated Diseases” (not financed); 3. FP7-Low Dose Research towards Multidisciplinary Integration (DoReMi): Epidemiologic study - systemic effects of low dose exposure to uranium on the immune status (not financed); 4. FP7 call Health-2013 Phosphodiesterases, inflammation endothelium dysfunction: a route to cardiovascular disease. Coordinator: Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Universite Libre de Bruxelles, , Brussels, Belgium (in preparation) Development plan I. New research areas: 1. Theranostics – Development of an integrated platform with fully 3D X-ray tomographic and microbeam fluorescence / luminescence guidance capabilities and of particular nanoparticles for simultaneous imaging and radiation therapy of tumors. Collaboration with the R&D Institute of for Laser, Plasma and Radiation Physics 2. Implementation of „omics” and high-throughput technologies for drug development and immunotoxicology (in collaboration with other teams from the institute) 3. Systemic effects of low dose exposure to ionizing radiation on the immune response; integration of the Radiobiology Laboratory in the European research network DoReMi (Integrating Low Dose Research) II. Patent submission for newly developed therapeutic compounds and design/screening of new ones III. Accreditation of new experimental methods relevant for drug development and toxicology IV. Introduction of new investigations in the Diagnosis Center (trace microelement profiling) V. Enlargement of national and international collaborations within IMI, FP7 etc - Focus on private companies VI. Increased exploitation of existent infrastructure, to be translated in an increased number of publications in ISI ranked journals. Development of Radiobiology laboratory’s specific infrastructure.
11 project proposals at the national 2011 Call “Partnerships – Collaborative projects of applied research” (under evaluation) Pathologic mechanisms and drug targets: 1. Fibro‐inflammatory biomarkers of early myocardial remodeling in heart failure patients with preserved ventricular ejection fraction–Elena MOLDOVEANU; 2. Optimization of autologous bone marrow stem cell therapy in patients with ischemic heart failure–Elena MOLDOVEANU 3. The importance of phosphodiesterase and thrombin in pulmonary fibrotic diseases‐ possible future therapeutic targets – Daciana MARTA. Drug development: 1. New chemicals and natural compounds for improving radiotherapy in cancer – radiosensitization, internal irradiation and radioprotection-Gina MANDA; 2. Translation of coordinative chemistry in biology and therapy. Complex compounds of physiological cations with thiosemicarbazones and tiocarbohyrdazones as antitumoral and/or antibacterial drugs–Mircea LEABU; 3. New epigenetic drug – DHA – in breast cancer therapy and biomarkers discovery–Sevinci POP; 4. Magnetic Nanostructures for Targeted Therapy – Sevinci POP; 5. Tool design for pharmacovigilance. Prediction of life-threatening angioedema for patients treated with renin-angiotensin system inhibitors and/or glyptins–Mircea LEABU. Toxicology: 1. Impact of feed co‐contamination and mitigating solutions to increase feed safety, animal health and food quality – Gina MANDA.
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TEAM 10 - Assay Development and Alternative Testing
Team members
First and Last Name Role Involvement Monica Neagu Team Leader 0,50 Carolina Constantin Member 0,50 Dan Ciotaru Member 0,3 Mihaela Surcel Member 0,3 Cristiana Tanase Member 0,30 Radu Albulescu Member 0,15 Elena Codrici Member 0,2 Daniela Popescu Member 0,25 Lucian Albulescu Member 0,2 Emilia Manole Member 0,3 Gheorghita Izvoranu Member 0,3 Alina Nita Member 0,2 Angela Petrescu Member 0,5 TOTAL 4,00
Technical personnel
First and Last Name Role Involvement Mariana Caralicea Technician 0.5 Georgiana Dumitrascu Technician 1 Mariana Pisica Technician 0.5 Irina Radu Technician 0,2 Laurentiu Anghelache Technician 0.5 TOTAL 2.7
In the last 4 years our team was involved in the following research directions: Immune-based assay development and Cell-based assays.
In the domain Immune-based assay development we are currently involved in innovative immune-detection in infectious diseases through the cooperation with University of Tubingen and University of Athens (NATO SfP 982838/2007). We have established a complete new testing approach for early bacterial infection testing. The innovative assay development consists in designing both new antibodies specific for C-terminal prothymosine peptide and fluorescence-immunoassay detection methods. In the last 4 years we have published the relation between the in vitro and in vivo bacterial infection and the release of a C-terminal prothymosine peptide. In biological fluids the developed immune-assay can detect in experimental mouse models the mentioned peptide as early as 2 hours post-infection. The possibility to detect as early as 2 hours post-infection a marker by means of chemiluminometric testing is a major breakthrough in this domain. The future of these results will be the development of an original international patent and the technological transfer for developing an easy-to-perform, sensitive and quick test for bio-terrorist attack. The research funds obtained in the framework of this NATO SfP 982838/2007 project are 300,000 Euros for Romania.
In the domain of Cell-based assays development we are developing efficient technology/workflow for drug potency identification with emphasis on nano-drugs for controlled delivery. In the last 4 years through a long lasting collaboration with the National Institute for
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Chemistry Bucharest we have developed several classes of photosensitizing compounds with anti-tumoral effect. Target potency, cytotoxicity, and metabolic liabilities were evaluated and the selected compounds entered the animal models testing. In animal models vivo efficacy was tested when reliable models were available. The hit-to-lead process induced a close collaboration between Assay Development Team and other teams, such as Proteomic and Biomarkers and Drug Development. When a promising lead series has been identified, besides publications, several composition-of-matter patents were accomplished and the internationally recognized value was recognized by awards. The patent OSIM Mr. 00489/25.06.2008 entitled “Tetra-sulphonated porphyrin application for producing a dermatologic therapy – photosensitizer” received Gold medal at Brussels Innova 2008, Special Prize of Rudy Demotte, Minister President of the Walloon Government, Gold medal at The 37th International Exhibition of Inventions of Geneva 2009 and Special Prize of the Ministry of Education of Rusia, 2009; Gold medal at The International Fair for Innovation, Moscow, 2009. Recognition of the last 10 years long-standing work performed by us in this domain, resides in our affiliation to the international networks: COST D39 Metallo-Drug Design & Action (2006-2010); COST TD1002 European network on applications …in NanoMedicine and Life Sciences (2011-2015). In the cell-based assay development we have developed specific equipments for cell imaging that were subject for patent OSIM A/00351/2019 / 21.04.2010 entitled “Equipment and procedure for microwave irradiation in in vitro models with concomitant registration of biological behaviour in a fluorescence microscope”. Developing several nationally granted projects the funds obtained by the research team in this domain heaves up to 850,000 Euros. Through cooperation with the University of Lisbon and Technical Institute of Portalegre, we have developed several classes of nano-compounds intended to be intracellular trackers in tumor cells and indicators for minimum residual disease in blood circulation. The project has already a patent OSIM Nr. RO-BOPI2/2009 entitled “Tetrapirolic compound asymmetrically substituted – synthesis and biological evaluation” and until 2012 it will develop several others in the cell-based assay for efficient drug delivery. The future of this research direction lays in increasing the nano-drug specificity with emphasis in targeting tumour receptors and tissue markers for not only a controlled delivery in time but as well in space. Through this MNT-ERA –NET 7050/2010 project the team was financed with 115,000 Euros. The involved laboratories are affiliated to the National Platform for Nanomedicine.
Related to the domain of cell-based assay the team leader is an active member of the Commission for Advanced Therapies – European Medicine Agency and one team member is evaluator for EuroNanoMed Projects.
Members of the Assay Development team are actively pear-reviewing for the following scientific journals: Pigments and Dyes, Photochemical and Photobiological Sciences, Patents in Biomarkers, Archives of Gerontology and Geriatrics, International Journal of Photoenergy, Romanian Biotechnological Letters, Romanian Archives of Microbiology and Immunology, Materials Science and Engineering B, Journal of Clinical Laboratory Analysis, International Journal of Nanomedicine. Two team members are also members of the editorial boards and invited editors for hot scientific topics in scientific journals such as Journal of Immunoassay&Immunochemistry and Recent Patents on Biomarkers.
The personnel dynamics in our team is remarkable, namely we have a mean age of 40.2 years and equilibrated between young researchers and more experienced senior researchers and have hosted through the international collaborations several PhD students that fulfilled their
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thesis in subject developed by the research team. The knowledge up-grading of our team is continuous such as, each of the team members attends international courses on up-to-date technologies. In 2007 the following courses and training stages were attended - Profiling Kinases and Phospho-Sites with Antibody-Based Methods for Disease Biomarker; Drug Target Discovery and Protein Arrays for Biomarker Discovery and Protein Expression Profiling, Amsterdam, Holland; In 2008: Principles and applications of microfluidics in the life sciences, Microfabrication technologies, Barcelona, Spain; ProteinChip SELDI-ToF MS Training Course, Malvern, USA; In vivo confocal microscopy training, Mavig, Bucharest, Romania; In 2009 - Flow Cytometer FACS CANTO II Training Course, Heildelberg, Germany; In 2010-2011 xCELLIgence Users Meeting, Munchen, Germany, Intensive Educational Course in Clinical Immunology, Centre de Recherche des Cordelieres, Paris; 2011 Course “Characterizing and applying physiologically-based pharmacokinetic models in risk assessment” – WHO Paris.
We have constant young Bachelor of Science degree personnel that perform their diplomas in the framework of the mentioned domains. The Laboratories involved are constantly hosting in PhD students or post-doctoral fellows in the framework of the mentioned international collaborations. The laboratories involved in the Assay development team have high-throughput technology with specialized software. Although the equipment is recently purchased (2007-2011) de level of exploitation by the described team is over 75%.
Domain Equipment 2D electrophoresis 2D-DIGE (Typhoon 9000) SELDI-ToF-MS Western blotting Multiplex xMAP® technology Protein microarray
Proteomics
Complete ELISA lines xCELLigence impedance measurement equipment Confocal microscopy Varioskan Multimode Reader for time-resolved fluorescence, chemiluminescence
Cellular physiology testing
Complete unit for cell culture manipulation and storage Appended publications to the team: A7, A27, A29, A31, A41, A49, A51, A55, A84, A91, A98 with a cumulated rAIS of 8,864 and 20 ISI citations Projects that funded the mentioned research directions CF3, CF4, CF12, CF13 have a total budget of 600,000 Euro and F27, F28, F29, F35, F43, F45, F46, F109, F110, F112, F113 850,000 Euro
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4. Representative project
TELOCYTES - A NEW TYPE OF INTERSTITIAL CELLS
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‘Victor Babeş’ National Institute of Pathology, Bucharest, Romania
TELOCYTE - A NEW TYPE OF INTERSTITIAL CELL
http://www.telocytes.com
KEY PERSONS INVOLVED IN TEOCYTE PROJECT
Team leader: Acad. Laurentiu M. POPESCU
Senior researchers: Hinescu ME, Ardeleanu C, Mandache E
PostDoc: Gherghiceanu M, Suciu LC, Popescu BO, Cismaşiu V, Cretoius S
PhD students: Manole CG, Nicolescu MI, Cretoiu D
INTERNATIONAL COLLABORATION • Prof. Maria-Simonetta Faussone-Pellegrini and Prof. Daniele Bani from Department of Anatomy,
Histology and Forensic Medicine, University of Florence, Italy • Prof. Sawa Kostin from Max-Planck Institute for Heart and Lung Research, Franz Groedel Institute,
Bad Nauheim, Germany • Prof. Shengshou Hu from the Center for Cardiovascular Regenerative Medicine, Peking Union
Medical College, Chinese Academy of Medical Sciences, Beijing, China • Prof. Changyong Wang from the Tissue Engineering Research Center, Academy of Military Medical
Sciences, Beijing, China TELOCYTES - A CASE OF SERENDIPITY: the winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to TELOCYTES The history of TC is recent since these cells have been discovered only a few years ago. However, the growth of knowledge on TC has been exponential from the beginning and we already have much information. TC were discovered in 2005 when L.M. Popescu’s group from Bucharest, Romania, described a new type of cell that resides in the stroma of several organs, which became known as interstitial Cajal-like cells (ICLC). This group named these cells ICLC because of their apparent similarity with the canonical gastrointestinal interstitial cells of Cajal (ICC), the gut pacemaker cells. A few years later, in 2008, M.S. Faussone-Pellegrini and her team from Florence, Italy, described ICLC in the muscle coat of the human gut and noticed they consistently differed from the ICC in both ultrastructure and immunophenotype. In 2010, after confirming the presence of this peculiar cell type in the stroma of many organs and characterized it by immunohistochemistry and electron microscopy, the two groups agreed they were describing a ‘novel’ cell type and that the name ICLC had to be changed with a more appropriate one. From then on, this novel cell type became known as the telocyte.
TELOCYTES - A NEW TYPE OF INTERSTITIAL CELLS
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TELOCYTES (TC) - NEW TYPE OF INTERSTITIAL CELLS WITH LONG PROLONGATIONS NAMED TELOPODES (Tp).
MORPHOLOGY OF TELOCYTES To characterize these cells, many different techniques have been used: in vitro, isolated cells in culture; in situ, observation on fixed specimens; light and fluorescence microscopy; transmission electron microscopy; scanning electron microscopy; electron tomography All these techniques have shown that TC are cells with a small body and a variable number of Tp. The shape of the cell body depends on the number of Tp and can be piriform/spindle/triangular/ stellate. The nucleus is oval, with a moderately dense chromatin, and has no obvious nucleolus. The cytoplasm surrounding the nucleus is scarce and contains a small Golgi apparatus, some mitochondria, and few cisternae of rough and smooth endoplasmic reticulum. Telocytes are certainly defined by their ultrastructural features. Usually, a TC has extremely long Tp with moniliform aspect generated by alternating podoms and podomers. Telopodes have particular characteristics:
1. Number : 1–5/cell, usually 1-3 2. Branching: dichotomous pattern 3. Length: tens up to hundred micrometres 4. Aspect moniliform - podomeres alternating with podoms 5. Podomers - 50-100 nm thin segments; usually < 0.2 μm, below the resolving power of light
microscopy 6. Podoms - dilated segments accommodating mitochondria, ER and caveolae (‘Ca2+ release units’) 7. Connected each other by junctions form an interstitial network
Immunohistochemistry To know the chemical code of TC is of fundamental importance since it allows their unequivocal identification and also helps evaluate their size, shape, number, and, eventually, movements, migration, and pathological changes. Although we made many reliable attempts testing an enormous variety of antibodies, a single marker that can be considered specific for this cell type or, at least, specific for the TC of a given organ has not been found. TC might show different immunohistochemical profiles among organs and even in the same organ examined. However, at present, CD34 labeling remains the best available choice for TC identification, possibly in combination with c-kit and vimentin labeling. Due to these important differences in TC immunolabelling and since none of the markers tested are ‘specific’, we need to solve this issue and perform further immunohistochemical techniques, including immunoelectron microscopy. Distribution TC have been found in a large variety of cavitary organs: [heart (endo-, myo-, and pericardium); stomach and intestine, with mesentery; gallbladder; uterus and Fallopian tube] and non-cavitary organs [lungs and pleura; pancreas (exocrine); mammary gland; placenta]. All the cells identified as TC were: - organized in a 3D network, dispersed in the extracellular substance, and intermingled with resident
(fibroblasts, mast cells, adipocytes) and nonresident (macrophages, immune cells, granulocytes) cells - localized at the connective border of various tissues (epithelial, muscular, and nerve tissues) lining them
and around blood vessels.
TELOCYTES - A NEW TYPE OF INTERSTITIAL CELLS
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ROLES OF TELOCYTES Several roles have been suggested for TC, most of which are believable and not mutually:
- key players in organ specific renewing (heart, lung, striated muscle) and could act as stromal support cells for stem cells. Ultrastructural analysis proved that TCs cardiac network could integrate the overall ‘information’ from vascular system (endothelial cells and pericytes), nervous system (Schwann cells), immune system (macrophages, mast cells), interstitium (fibroblasts, extracellular matrix), stem cells, progenitors and working cardiomyocytes. Generally, heterocellular contacts occur by means of minute junctions (point contacts, nanocontacts and plane contacts) and the mean intermembrane distance is often within the range of tens of nm (10-30 nm) which fits in the molecular interactions domain. Our study showed that homotropic and heterotropic ultrastructural interactions of TCs in adult heart form an integrative interstitial system. Possibly, TCs network assure physiological coordination of multicellular signals, essential for stem cells (resident or circulating) decision to proliferate, differentiate and mature into new cardiomyocytes or other cardiac cell types.
- sustain myocardial tissue organization in developing and adult heart - in immune surveillance (stromal synapse with mononuclear cells, granulocytes, mast cells,
macrophages) - TC could be mesenchymal stem cells (MSC); in vivo identity of MSC is still unknown (TC and MSC
are CD34+ cells) - tensional integration of the tissue, considering their characteristic ultrastructure (extremely long and
contorted processes with intermediate filaments and microtubules parallel to the long axis of the cell, attachment plaques connecting it to the extracellular matrix)
- in neurotransmission in the gut, possibly contributing to spread the slow waves generated by the ICC.
Even the TC are not fully characterized and their roles are speculative, recent studies showed that TC may be involved in a few important pathologies:
- isolated atrial amyloidosis and atrial fibrillation; - neoangiogenesis in cardiac recovery after experimental myocardial infarction; - PEComas -perivascular epithelioid cell tumours; - GISTs-gastro-intestinal and extra-gastrointestinal stromal tumours.
Funding from research grants: NUCLEU PN 06.26/2005-2008; CEEX 112/2006-2008; NUCLEU PN PN09_33/2009-2011 FUTURE RESEARCH DIRECTIONS
- Telocytes - specific markers - Telocytes - origin and lineage tracing - Role of telocytes in physiology and pathology
ANNEX 1 - Reprint of “Faussone-Pellegrini MS, Popescu LM (2011) TELOCYTES. BioMolecular Concepts DOI: 10.1515/BMC.2011.039”
ANNEX 2 - Reprint of “Popescu LM, Faussone-Pellegrini MS (2010) TELOCYTES – a case of serendipity: the winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to TELOCYTES. J Cell Mol Med 14: 729–740 (24 ISI citations)
TELOCYTES - A NEW TYPE OF INTERSTITIAL CELLS
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ONGOING PROJECT: ‘TELOCYTES IN HEART RENEWAL’ project coordinated by Acad. L.M. Popescu (348.837 euro) IDEI-PCE 2011 competition for 3 years (2011-2014) Stem cell therapy for cardiac diseases has been started before an intrinsic regenerative capacity of heart to be proved and accepted. The dogma that mammalian heart is terminally differentiated organ has been challenged by the reports of few types of resident cardiac stem or progenitor cells. Moreover, a new type of interstitial cell – telocyte - has been described in the adult heart and one important role seems to be nursing stem cells and progenitors in the cardiac stem cell niches. We plan to study the cellular and developmental biology of cardiac stem niches and their involvement in cardiac renewal considering that basic mechanisms governing its physiology are still unknown. By extensive ultrastructural investigation (electron tomography included), confocal microscopy and miRNAs detection we plan a basic research of telocytes and cardiac stem cell niches in normal, ageing and diseased mammalian heart. We also will run a comparative study of regeneration in mammalian and zebrafish (known to have high regenerative capacity) injured hearts. We will try to answer major question: there are one or more types of cardiac stem cell; which cells are mandatory for cardiac renewal; which factors are most important in stem cell differentiation; how newly formed cardiomyocytes are integrated in contractile myocardium; how all these are challenged in diseased heart? All these questions must be answered before an effective cell therapy could be envisaged.
TELOCYTES - A NEW TYPE OF INTERSTITIAL CELLS
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Visibility
BioMol Concepts, Vol. xx (2011), pp. xxx-xxx • Copyright � by Walter de Gruyter • Berlin • Boston. DOI 10.1515/BMC.2011.039
2011/0038
Article in press - uncorrected proof
Review
Telocytes
Maria-Simonetta Faussone Pellegrini1,* andLaurentiu M. Popescu2,3¸1 Section of Histology, Department of Anatomy, Histologyand Forensic Medicine, University of Florence, Viale G.Pieraccini 6, I-50139 Florence, Italy2 Department of Cellular and Molecular Medicine ‘CarolDavila’ University of Medicine and Pharmacy, Bucharest,Romania3 Department of Advanced Studies, ‘Victor Babes’ NationalInstitute of Pathology, 99-101 Splaiul Independentei,¸RO-050096 Bucharest, Romania
* Corresponding authore-mail: [email protected]
Abstract
Here, we review the history, morphology, immunohistoche-mical phenotype, and presumptive roles of a new type ofinterstitial tissue cells, formerly called interstitial Cajal-likecells (ICLC) and by 2010 named ‘telocytes’ (TC). Manydifferent techniques have been used to characterize TC andprovide their unequivocal identification: (i) in vitro, culturesand isolated cells; (ii) in situ, fixed specimens examined bylight and fluorescence microscopy, transmission (TEM) andscanning electron microscopy, and electron tomography.TEM allowed sure identification and characterization of themost peculiar feature of TC: the long, thin, and convolutedprolongations named ‘telopodes’. An enormous variety ofantibodies have been tested, but presently none are reliableto specifically label TC. TC have a mesenchymal origin andare resident connective tissue (stromal) cells. Possible iden-tification with ‘already identified’ stromal cell types (fibro-blasts, fibrocytes, fibroblast-like cells, and mesenchymalstromal cells) is discussed. We conclude that in adulthood,most of the TC have the morphology of fibrocytes. Appar-ently, immunocytochemistry suggests that a variety of TCpopulations showing different, likely organ-specific, immu-nophenotypes might exist. Several roles have been hypoth-esized for TC: mechanical roles, intercellular signaling,guiding and nursing of immature cells during organogenesis,and being themselves a pool of precursors for many of themesenchyme-derived cells in adulthood; however, none ofthese roles have been proven yet. On the basis of the avail-able data, we propose TC may be key players in organ regen-eration and repair.
Keywords: fibroblasts, interstitial Cajal-like cells (ICLC),interstitial cells of Cajal (ICC), mesenchymal cells,telopodes.
Introduction: a short history
The history of telocytes (TC) is recent since these cells havebeen discovered only a few years ago. However, the growthof knowledge on TC has been exponential from the begin-ning and we already have much information. TC were dis-covered in 2005 when L.M. Popescu’s group from Bucharest,Romania, described a new type of cell that resides in thestroma of several organs (1–7), which became known asinterstitial Cajal-like cells (ICLC). This group named thesecells ICLC because of their apparent similarity with thecanonical gastrointestinal interstitial cells of Cajal (ICC), thegut pacemaker cells (8–11). A few years later, in 2008, M.S.Faussone-Pellegrini and her team from Florence, Italy,described ICLC in the muscle coat of the human gut andnoticed they consistently differed from the ICC in bothultrastructure and immunophenotype (12). In 2010, afterconfirming the presence of this peculiar cell type in the stro-ma of many organs and characterized it by immunohisto-chemistry and electron microscopy, the two groups agreedthey were describing a ‘novel’ cell type and that the nameICLC had to be changed with a more appropriate one (13).From then on, this novel cell type became known as thetelocyte (13).
Rationale for the term ‘telocyte’
The interstitial tissue making up the stroma of an organ isthe connecting ‘device’ for the specific structures of theorgan and the resident connective tissue cells are usuallynamed ‘stromal cells’. However, according to some authorsand depending on the organ where these cells reside, theyhave received a variety of other names: fibroblasts, fibro-cytes, fibroblast-like cells, myofibroblasts, mesenchymalcells, interstitial cells, and ICLC. Under transmission elec-tron microscopic (TEM) examination, the cells formerlycalled ICLC reveal all their characteristics that are unique,unequivocal, and not yet described for any other cell type.To avoid further confusion and to give a precise identity tothese cells, Bucharest’s team coined for them the term ‘telo-cyte’ wscell bearing long prolongations (13)x on the basis oftheir most peculiar feature: the presence of prolongations thatare extremely long (tens to up to hundreds of micrometers,as measured on TEM images), thin (mostly below 0.2 mm),and with a moniliform aspect (Figures 1–5). The concept ofTC was promptly adopted by this group and several otherlaboratories (14–24).
Q1:Pleasesupply thepostal codefor affiliation2
Q2:Pleasesupply ashort generaltitle/captionfor Figures 1and 4
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2 M.-S.F. Pellegrini and L.M. Popescu
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Figure 1 (A and B) CD34-immunoreactivity; submucosa of human stomach. (A) The CD34-positive cells (in brown) have a triangular orovoid body and a variable number of thin and long prolongations that give a stellate shape to the whole cell. These processes have knobsalong their length and a dichotomous branching. Bar, 20 mm. wWith permission from (12).x (B) A detail of the body and prolongations ofa CD34-positive cell. Bar, 15 mm. (C) Human nonpregnant myometrium in cell culture, day 3, first passage. Giemsa staining. One telocyteestablishing contact with a smooth muscle cell (myocyte) by a cell process (telopode) of about 65 mm long. Photographic composition offour serial phase contrast images; original magnification, 40=. In the red rectangles, a higher magnification clearly shows the moniliformaspect of the telopode; at least 40 specific dilations (podoms) of the telopode, interconnected by thin segments (podomeres), are visible ina ‘beadlike’ fashion. Original magnification, 40= wWith permission from (3).x (D) Human resting mammary gland stroma, TEM. Onetelopode, which appears very long and convoluted, with intercalated podomeres and podoms. Note the homocellular junctions marked byred circles, as well as shed vesicles (SV, blue) and an exosome (violet). coll, collagen. wReproduced with permission from (1).x
Figure 2 TEM imaging.(A) Rat mesentery. One TC with a small nucleated body and three long telopodes, all of them having a sinuous trajectory and forming aninterstitial complicated 3D network. (B) Human exocrine pancreas. The TC form with their typical long telopodes a network around theacini.
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Telocytes 3
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Figure 3 Electron tomography (thick section of about 300 nm)showing nanostructures connecting one telopode with two adjacentcardiomyocytes in adult mouse heart.The bridging structures (encircled) are 10–15 nm and suggest amolecular interaction between the telopode and the cardiomyocytes.The dilated segment of this telopode contains a mitochondrion (m).wReproduced with permission from (25).x
Figure 4 (A) Human stomach. Double CD34/c-kit labeling. CD34 positivity is represented in red and c-kit positivity in green. The CD34-and c-kit-positive cells are often very close to each other but none of them are double labeled. Bar, 25 mm. (B and C) CD34 immunoelec-trolabeling. (B) Mouse small intestine. CD34 positivity is present along the plasma membrane of a long, thin process of a TC, while ICC,nerve fibers, and smooth muscle cells (SMC) are CD34 negative. Bar, 1 mm. (C) Mouse stomach. Detail of a CD34 immunoelectrolabeledtelopode. CD34 positivity clearly appears as electron-dense spherules regularly distributed on the telopode plasma membrane. Bar, 0.4 mm.
Morphology
To characterize these cells, many different techniques havebeen used: (i) in vitro, isolated cells in culture (Figure 1C);(ii) in situ, observation on fixed specimens; (iii) light (Figure1A,B) and fluorescence microscopy (Figure 4A); (iv) trans-mission electron microscopy (Figures 1D, 2A,B, 4B–D and5); (v) scanning electron microscopy; (vi) electron tomog-raphy (Figure 3).
All these techniques have shown that TC are cells with asmall body and a variable number of long prolongationsnamed telopodes (Tp). The shape of the cell body dependson the number of Tp and can be piriform/spindle/triangular/stellate (Figures 1A,B, 2A,B and 4A). The nucleus is oval,with a moderately dense chromatin, and has no obviousnucleolus. The cytoplasm surrounding the nucleus is scarceand contains a small Golgi apparatus, some mitochondria,and few cisternae of rough and smooth endoplasmic reticu-lum (Figure 2A,B). Average dimensions of the TC body are9.3"3.2 mm (min. 6.3 mm; max. 16.4 mm). Mitochondriarepresent 2% of cell body volume. Each TC can have1–5 Tp. However, frequently only 2–3 Tp are observed ona single section depending on site and angle of the sectionsince their three-dimensional (3D) convolutions prevent themfrom being observed at their full length in a 2D thin section.Convolutions of the Tp, however, are not always present andhave variable extent and complexity depending on the organwhere TC are located. The Tp moniliform aspect (Figure1C,D) is due to an alternation of thin segments, podomeres(whose caliber is below the resolving power of light micro-scopy, 0.1"0.05 mm; min. 0.003 mm, max. 0.24 mm) anddilated segments, podoms, which accommodate mitochon-dria, rough and smooth endoplasmic reticulum, andcaveolae – the so-called Ca2q uptake/release units. Tp estab-lish several types of homo- and heterocellular junctions (Fig-ure 3) (25), release shed vesicles and exosomes (Figure 1D)(26), have a dichotomous branching pattern forming a 3D
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Figure 5 Schema and electron micrograph illustrating the relation of several telocytes (blue) with a column of cardiomyocyte progenitors(CMP, brown) in an epicardial stem cell niche of adult mouse.The telopodes run parallel to the main axis of the CMP column and seem to establish the direction of development. wReproduced withpermission from (27).x
labyrinthine network (Figures 1A and 2A), and might showadhesion plaques with the extracellular matrix (27).
Electron microscopy
Scanning electron microscopy provides good images of pre-sumptive TC (13), and electron tomography provides imagesof cell-to-cell contacts (25). However, TEM alone allowssure identification of TC, evaluation of the cell-to-cell inter-relationships, and a detailed description of the Tp.
Vital staining, cultures
In cell culture, TC shape and, in particular, Tp length can beeasily evaluated since in these conditions Tp are not con-voluted (Figure 1C). Moreover, MitoTracker Green FM, alipophylic selective dye that becomes fluorescent once itaccumulates in the lipid environment of mitochondria, con-firms these organelles are present in the TC body and at thelevel of Tp dilations (5, 28). Immunohistochemistry per-formed on cultured or isolated TC from determined organsdoes not always give the same results as those obtained onthe corresponding TC in situ.
Immunohistochemistry
To know the chemical code of TC is of fundamental impor-tance since it allows their unequivocal identification and alsohelps evaluate their size, shape, number, and, eventually,movements, migration, and pathological changes. Unfortu-nately, although Bucharest’s group made many reliableattempts in testing an enormous variety of antibodies (14,29, 30), a single marker that can be considered specific forthis cell type or, at least, specific for the TC of a given organhas not been found. Indeed, TC might show different immu-nohistochemical profiles among organs and even in the sameorgan examined. In the chorial villi (14), some TC are c-kitpositive and some CD34 positive; all of the CD34-positiveTC express vimentin and caveolin-1, and some of them alsoc-kit (14). In cultured cells from the same placental villi,some TC are double positive for c-kit and iNOS and othersfor c-kit and VEGF (14). Skeletal muscle TC are c-kit,caveolin-1, and CD34 positive, and have been found tosecrete VEGF (18). In the mouse heart, most of the TC areCD34 positive and a few are c-kit positive (31). Presently,only one study, performed by Florence’s group on both light/fluorescence and ultrastructural detection of CD34 and c-kitin the human gut, is available (12). In this study, immuno-
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Table 1 TC distribution.
Cavitary organs Noncavitary organs
Heart (endo-, myo-, and pericardium)(6, 16, 19, 20, 25) (26–31) Lung and pleura (17)Stomach and intestine (12, 21) Exocrine pancreas (2)Gallbladder (32) Mammary gland (1)Uterus (3, 33, 34) Placenta (14)Fallopian tube (5, 35) Skeletal muscle (18)Blood vessels (36) Mesentery (37)
histochemistry combined with TEM allowed to put into lightthat the enteric TC are CD34-positive and c-kit-negativecells, and also to exclude ICC and TC as being the same celltype (Figure 4A,B,D). Notably, the TC located in the mucosaare neither CD34 nor c-kit positive (12). Briefly, CD34 label-ing does not allow an unequivocal TC identification since itdoes not label all TC, at least in some organs we.g., gut,placenta, and striated muscle (12, 14, 18)x or during embry-onic life we.g., in the heart (20) and gut (Faussone-Pellegrini,personal observations)x. However, at present, CD34 labelingremains the best available choice for TC identification, pos-sibly in combination with c-kit and vimentin labeling.
In conclusion, due to these important differences in TCimmunolabeling and since none of the markers tested are‘specific’, it would be desirable if more laboratories otherthan those in Bucharest and Florence will study this issueand perform further immunohistochemical techniques,including immunoelectron microscopy.
Distribution
TC have been found in a large variety of cavitary and non-cavitary organs (Table 1). Therefore, we would reasonablyconclude these cells are ubiquitous. All the cells identifiedas TC were located within the connective tissue and couldbe (i) organized in a 3D network, dispersed in the extracel-lular substance, and intermingled with resident (fibroblasts,mast cells, adipocytes) and nonresident (macrophages,immune cells, granulocytes) cells, or (2) at the connectiveborder of various tissues (epithelial, muscular, and nerve tis-sues) lining them and around blood vessels; these TC arelikely organized in a 2D network. Those located aroundblood vessels presumably correspond to the adventitial cellsand those located around myenteric plexus ganglia and nervestrands correspond to the covering cells (12, 38).
Possible TC identification with ‘already
identified’ stromal cell types
Usually, connective tissue cells are perceived as being mainly(or even only) fibroblasts and/or fibrocytes. Importantly, ithas to be noted that across different countries and laborato-ries, there is great confusion between the terms fibroblast andfibrocyte. In some European countries and outside of Europe,only the term fibroblasts is used; however, two types of
fibroblasts are recognized, the active and the quiescent fibro-blasts. Conversely, in some other European countries (Italy,Germany, Romania, etc.) both fibroblasts and fibrocytes arerecognized as distinct cell types. There is a general agree-ment that cells called fibroblasts correspond to the activeconnective tissue cells involved in synthesis and organizationof extracellular components (ground substance and fibers).These cells are, therefore, obviously present during devel-opment and whenever there is a need for renewal or repairof extracellular components. The so-called fibrocytes con-ceivably correspond to the quiescent fibroblasts, which aretypical of connective tissues during adult life. The distinctionbetween fibrocytes and fibroblasts is based on their markedlydifferent ultrastructural features; these differences are com-monly reported in histology textbooks and can be summa-rized as follows.
The (active) fibroblast body is large and pleomorphic; thenucleus is typically euchromatic and has one to two nucleoli;the Golgi complex is prominent; and the rough endoplasmicreticulum is well developed (about 5–12% of cell volume).Cell processes are few, short, and of large caliber, thus beingeasily appreciable under a light microscope. These cells aremarkedly different from TC, but some of the cells labeledas ‘fibroblasts’ in the figures reported in histology textbooksand other literature show the morphology of TC and not thatof active fibroblasts. Recently, some markers have been test-ed to differentiate cardiac TC from fibroblasts (which arec-kit negative) (39). Noteworthy, microRNA expression(e.g., miR-193) clearly differentiated TC from fibroblasts andin culture also allowed to discriminate between TC and otherstromal cells (39).
The body of fibrocyte (or quiescent fibroblast) cells issmall and oval; the nucleus is moderately heterochromaticand the nucleolus is difficult to locate; the Golgi complex issmall; and the rough endoplasmic reticulum is scarce. Cellprolongations are few, long, and thin (usually described astapering, slender processes). Intriguingly, the body, nucleus,and some of the ultrastructural characteristics of fibrocytesare the same as those reported for the TC, with the exceptionof the extension, convolution, and moniliform aspect of pro-cesses. The morphology of the cells labeled as ‘fibrocytes’in figures reported in histology textbooks and other literatureis very similar to that of the TC. In figure 1 of a recentreview (40), the cells located in the connective tissue havethe typical features of both fibrocytes and TC. These lattercells are named fibrocytes and considered to be circulatingmesenchymal progenitor cells that participate in tissueresponses to injury and invasion (40).
Cells having the ultrastructure of active fibroblasts havebeen described both in normal and pathological conditionsby many laboratories, but there is a general agreement thatthey are not to be considered as true fibroblasts: thus, thesecells were named fibroblast-like cells. No specific functionhas been attributed to them. These cells do not resemble TC.
According to histologists and pathologists, mesenchymalstromal cells are cells still present in adulthood that are most-ly arranged along blood vessels, particularly along capillaries(41). In usual histology textbooks, these cells are never
Q3:Please checkthe edits inthe sentence‘Accordingtohistologists...’
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shown, but their description corresponds to that of smallfibroblasts. On the contrary, embryonic mesenchymal cellsare shown in many embryology textbooks and other litera-ture. These cells have a round body filled with free ribo-somes, a small Golgi complex, a nucleus with clearchromatin, and a large nucleolus. Cell processes can beabsent, or when present are usually long and thin. TCmorphology, therefore, does not correspond to that of mes-enchymal cells, at least not those described during embryoniclife.
Pre- and postnatal differentiation
Studies aimed at gaining information on pre- and postnatalTC differentiation have not been performed, and little infor-mation is currently available. Such studies would give ananswer on which is the origin of TC and on whether thesecells are differentiated or immature cells. The knowledge ofTC morphology and immunophenotype during their matu-rative steps could be of great help in identifying all TC var-iations during development and in recognizing them at everyage. Noteworthy, it is possible that, according to the organwhere they reside, TC could show different degrees of dif-ferentiation. This possibility is suggestive for the presence ofTC subtypes in adulthood, which differ from each other inmorphology, immunophenotype, and name. Also, studiesaimed at investigating whether TC can retain the capabilityto further differentiate, are committed to one or more specificcell lineages, and are able to differentiate spontaneously orafter injury, would be welcome.
At present, Florence’s team indirectly provided some evi-dences for TC differentiation. Results obtained by studyingthe developing mouse heart, as well as primary cultures ofneonatal mouse cardiac cells (20, 42), showed that at earlierembryonic stages wembryonic day 14 (E14)x, all the intersti-tial cells had typical mesenchymal ultrastructural featuresand none of them were CD34 positive. However, by E17,some of them acquired CD34 positivity and, at birth (P0),also fibroblast-like features. After birth (by P6), the putativeTC showed their typical ultrastructural features while CD34positivity became uncertain or limited to few of them. Ofnote, the acquisition of the immuno- and the ultrastructuralphenotypes are not synchronized. In studies on the humansmall intestine from fetal life to birth and on ICC plasticity(43, 44), mesenchymal cells were seen to become ICC andsmooth muscle cells passing through an intermediate celltype having fibroblast-like features. TC precursors also havefibroblast-like features; however, at variance with the ICCthat were c-kit positive and already differentiated in fetusesat term (45), TC are never c-kit positive and acquire theirtypical CD34 positivity and ultrastructure only after birth(Faussone-Pellegrini, unpublished data). According to somerecent data (46, 47), the CD34-positive cells present in thesefetuses might be in fact immature ICC.
On the basis of the available information, we can reason-ably conclude that TC are mesenchymal in origin, are resi-dent connective tissue cells from their earliest developmental
steps, and during their differentiation share fibroblast-likefeatures with immature ICC, smooth muscle cells, and truefibroblasts. An obvious question arises when studying TCdifferentiation: does the TC correspond to fibroblast-likecells? In adulthood, most if not all of the TC have the mor-phology of cells called fibrocytes; however, presently wehave no information on whether different TC populationsexist in the various organs and on whether some of themmaintain fibroblastic-like appearance also in adulthood.Moreover, it cannot be excluded that TC will acquire thisfeature in the presence of stimuli to tissue renewal or repaircontributing to (i) new synthesis of the extracellular com-ponents, or (ii) differentiation of new cells to replace thedead ones, or (iii) spatial reorganization of the organ.
We would like to hypothesize TC are ‘progenitor cells’more or less committed according to the organs and still ableto further differentiate. This hypothesis opens a wide andfascinating field for future researches that will surely provideresults that are surprising and of high impact.
Roles
Several roles have been suggested for TC, most of which arebelievable and not mutually exclusive. However, none ofthem have been proven yet.
TC might have a role as a mechanical support. The TC ofthe rat mesentery form a 3D network hypothesized to be atthe same time resistant and deformable following stretchesconsequent to gut movements, mainly directed to avoidblood vessel closure (37). The TC located in the gut musclecoat, in particular those at the myenteric plexus level, alsoform a 3D network that is likely resistant to and deformablefollowing intestinal movements (12).
TC might guide the migration of other cells to define thefinal organization of an organ or its repair or renewal.According to a recent study (48), TC guide the migration ofmesenchymal cells into the mesothelial layer of the epicar-dium, thus being involved in mesothelial renewal. CardiacTC should guide myocardial precursors to form the correct3D tissue pattern and contribute to compaction of the embry-onic myocardial trabeculae. Indeed, cardiomyoblasts and TCwere seen to form stem cell (SC) niches in the subepicardialregion of the adult mouse heart (Figure 5) (27, 42, 49), tomigrate during development from the epicardium, fromwhere they presumably originated, and to form an extendednetwork of Tp closely embracing the growing cardiomyo-cytes (20). Results obtained in studies of co-cultures of TCand cardiomyoblasts confirmed that TC can intervene in theaggregation of cardiomyocyte clusters (20).
An immune surveillance role was suggested for the net-work of CD34-positive interstitial cells, further identified asTC, located in human fallopian tube (50).
Intriguingly, it has also been suggested that TC might playa role in neurotransmission in the gut, possibly contributingto spread the slow waves generated by the ICC. Indeed, theintramuscular ICC and TC seem to be part of a unique net-work, in which, however, only ICC are innervated (12).
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TC might be involved in intercellular signaling. The car-diac TC have been hypothesized to play a nursing role (27)and those in the oviduct and myometrium to be sensors forsteroid hormones (34, 35). Significantly, the Tp establishhomo- and heterocellular junctions (25); release shed vesi-cles and exosomes (26); and show paracrine secretion of IL6,VEGF, and NO. Thus TC, by sending macromolecular sig-nals to neighboring cells, could influence their transcriptionalactivity.
Finally, TC might represent a pool of cell precursors fora variety of cell types with common mesenchymal origin.Up to now, this role has been proposed for the placental (14)and enteric TC (12). The latter might be ICC precursors thatrenew ICC undergoing apoptosis (51), thus keeping the ICCnumber constant throughout life. Recently, a nonsatellite res-ident progenitor cell niche (presumably made by TC) wasdescribed in the striated muscle (18). In cultures of this tis-sue, TC (but not satellite cells) were seen to emerge frommuscle explants and form cell networks, suggesting a keyrole in muscle regeneration and repair (18).
Pathology
Information on pathological TC would be of high interest.At present, only one published paper deals with TC involve-ment in heart amyloidosis in patients with atrial fibrillation(52). By TEM, amyloid deposits were located in interstitialrecesses surrounded by long and slender TC processes, likelylimiting the spread of deposits into the interstitium. Intersti-tial cells, likely TC, have been characterized in the upperlamina propria of bladders of patients with neurogenic detru-sor overactivity and bladder pain syndrome. These cells wereseen to shift toward a fibroblast phenotype (53).
The study of TC in mutant animals is also a tantalizingchallenge. Presently, the only available information indicatesthe absence of caveolae in the TC from the myocardium (54)and gut muscle coat (55) of Cav-1 knockout mice. This find-ing remains unexplained, mostly because of the lack of phys-iological data.
Perspectives: regenerative medicine
It is tempting to speculate that TC, as progenitor and/or guid-ing and nursing cells, are a novel, possible target for thera-peutic strategies (56–58). The challenge of using muscleprogenitor cells for skeletal and cardiac muscle reconstruc-tion in animal models or humans has not been solved to date,mainly due to scarce graft cell survival explained by a lackof adequate paracrine factors, tissue guidance, and bloodvessel scaffold (59–64). Therefore, new attempts aimed atpotentiating cardiac repair and regeneration after ischemicinjury received great momentum from the hypothesis of acoexistence and cooperation of organ-specific TC and SC(20). Briefly, TC and SC can be seen as working in tandem,representing a better option for therapy rather than SC alone(65). An important goal would be to ascertain whether such
TC–SC cooperation requires homologous TC and SC fromthe same organ or whether TC from any organ source canbe used for cell therapy. In the first case, TC of a given organshould be committed to differentiate or cooperate with thecells specific to this organ, likely because of their similarembryologic origin (e.g., from the epicardium, the mesothe-lium, or even the endoderma, ectoderma and both intra- andextraembryonic mesoderma). In the second case, TC extract-ed from any organ could correctly function even when graft-ed into organs of a different embryologic origin, withobvious advantages in terms of availability and plasticity.
For more information on telocytes, see the papers andimages at http://www.telocytes.com.
References
1. Gherghiceanu M, Popescu LM. Interstitial Cajal-like cells(ICLC) in human resting mammary gland stroma. Transmissionelectron microscope (TEM) identification. J Cell Mol Med2005; 9: 893–910.
2. Popescu LM, Hinescu ME, Ionescu N, Ciontea SM, Cretoiu D,Ardeleanu C. Interstitial cells of Cajal in pancreas. J Cell MolMed 2005; 9: 169–90.
3. Ciontea SM, Radu E, Regalia T, Ceafalan L, Cretoiu D, Gherg-hiceanu M, Braga RI, Malincenco M, Zagrean L, Hinescu ME,Popescu LM. C-kit immunopositive interstitial cells (Cajal-type) in human myometrium. J Cell Mol Med 2005; 9: 407–20.
4. Popescu LM, Gherghiceanu M, Cretoiu D, Radu E. The con-nective connection: interstitial cells of Cajal (ICC) and ICC-like cells establish synapses with immunoreactive cells.Electron microscope study in situ. J Cell Mol Med 2005; 9:714–30.
5. Popescu LM, Ciontea SM, Cretoiu D, Hinescu ME, Radu E,Ionescu N, Ceausu M, Gherghiceanu M, Braga RI, Vasilescu F,Zagrean L, Ardeleanu C. Novel type of interstitial cell (Cajal-like) in human fallopian tube. J Cell Mol Med 2005; 9:479–523.
6. Hinescu ME, Popescu LM. Interstitial Cajal-like cells (ICLC)in human atrial myocardium. J Cell Mol Med 2005; 9: 972–5.
7. Radu E, Regalia T, Ceafalan L, Andrei F, Cretoiu D, PopescuLM. Cajal-type cells from human mammary gland stroma: phe-notype characteristics in cell culture. J Cell Mol Med 2005; 9:748–52.
8. Faussone-Pellegrini MS, Cortesini C, Romagnoli P. Sull’ultra-struttura della tunica muscolare della porzione cardiale dell’e-sofago e dello stomaco umano con particolare riferimento allecosidette cellule interstiziali del Cajal. Arch It Anat Embriol1977; 82: 157–77.
9. Thuneberg L. Interstitial cells of Cajal: intestinal pacemakercells? Adv Anat Embryol Cell Biol 1982; 71: 1–130.
10. Faussone-Pellegrini MS. Histogenesis, structure and relation-ships of interstitial cells of Cajal (ICC): from morphology tofunctional interpretation. Eur J Morphol 1992; 30: 37–48.
11. Faussone-Pellegrini MS. Interstitial cells of Cajal: once negli-gible players, now blazing protagonists. Ital J Anat Embryol2005; 110: 11–31.
12. Pieri L, Vannucchi MG, Faussone-Pellegrini MS. Histochemi-cal and ultrastructural characteristics of an interstitial cell typedifferent from ICC and resident in the muscle coat of humangut. J Cell Mol Med 2008; 12: 1944–55.
Page 8 of 16
8 M.-S.F. Pellegrini and L.M. Popescu
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13. Popescu LM, Faussone-Pellegrini MS. TELOCYTES – a caseof serendipity: the winding way from interstitial cells of Cajal(ICC), via interstitial Cajal-like cells (ICLC) to TELOCYTES.J Cell Mol Med 2010; 14: 729–40.
14. Suciu L, Popescu LM, Gherghiceanu M, Regalia T, NicolescuM, Hinescu M, Faussone-Pellegrini MS. Telocytes in humanterm placenta: morphology and phenotype. Cells Tissue Org2010; 192: 325–39.
15. Popescu LM, Manole CG, Gherghiceanu M, Ardelean A, Nico-lescu MI, Hinescu ME, Kostin S. Telocytes in human epicar-dium. J Cell Mol Med 2010; 14: 2085–93.
16. Gherghiceanu M, Manole CG, Popescu LM. Telocytes in endo-cardium: electron microscope evidence. J Cell Mol Med 2010;14: 2330–4.
17. Hinescu ME, Gherghiceanu M, Suciu L, Popescu LM. Telo-cytes in pleura: two- and three-dimensional imaging by trans-mission electron microscopy. Cell Tissue Res 2010; 343:389–97.
18. Popescu LM, Manole E, Serboiu CS, Manole CG, Suciu LC,¸Gherghiceanu M, Popescu BO. Identification of telocytes inskeletal muscle interstitium: implication for muscle regenera-tion. J Cell Mol Med 2011; 15: 1379–92.
19. Kostin S. Myocardial telocytes: a specific new cellular entity.J Cell Mol Med 2010; 14: 1917–21.
20. Bani D, Formigli L, Gherghiceanu M, Faussone-Pellegrini MS.Telocytes as supporting cells for myocardial tissue organizationin developing and adult heart. J Cell Mol Med 2010; 14:2531–8.
21. Carmona IC, Bartolome MJ, Escribano CJ. Identification oftelocytes in the lamina propria of rat duodenum: transmissionelectron microscopy. J Cell Mol Med 2011; 15: 26–30.
22. Cantarero CI, Luesma BMJ, Junquera EC. The primary ciliumof telocytes in the vasculature: electron microscope imaging. JCell Mol Med 2011; Mar 24. doi: 10.1111/j.1582-4934.2011.01312.x.
23. Zhou J, Zhang Y, Wen X, Cao J, Li D, Lin Q, Wang H, Liu Z,Duan C, Wu K, Wang C. Telocytes accompanying cardiomyo-cyte in primary culture: two- and three-dimensional cultureenvironment. J Cell Mol Med 2010; 14: 2641–5.
24. Eyden B, Curry A, Wang G. Stromal cells in the human gutshow ultrastructural features of fibroblasts and smooth musclecells but not myofibroblasts. J Cell Mol Med 2010; Jul 21.doi:10.1111/j.1582-4934.2010.01132.x.
25. Gherghiceanu M, Popescu LM. Heterocellular communicationin the heart: electron tomography of telocyte-myocyte junc-tions. J Cell Mol Med 2011; 15: 1005–11.
26. Mandache E, Popescu LM, Gherghiceanu M. Myocardial inter-stitial Cajal-like cells (ICLC) and their nanostructural relation-ships with intercalated discs: shed vesicles as intermediates. JCell Mol Med 2007; 11: 1175–84.
27. Popescu LM, Gherghiceanu M, Manole GC, Faussone-Pelle-grini MS. Cardiac renewing: interstitial Cajal-like cells nursecardiomyocyte progenitors in epicardial stem cell niches. J CellMol Med 2009; 13: 866–86.
28. Popescu LM, Gherghiceanu M, Hinescu ME, Cretoiu D, Cea-falan L, Regalia T, Popescu AC, Ardeleanu C, Mandache E.Insights into the interstitium of ventricular myocardium: inter-stitial Cajal-like cells (ICLC). J Cell Mol Med 2006; 10:429–58.
29. Hinescu ME, Gherghiceanu M, Mandache E, Ciontea SM,Popescu LM. Interstitial Cajal-like cells (ICLC) in atrial myo-cardium: ultrastructural and immunohistochemical characteri-zation. J Cell Mol Med 2006; 10: 243–57.
30. Kostin S, Popescu LM. A distinct type of cell in myocardium:interstitial Cajal-like cells (ICLCs). J Cell Mol Med 2009; 13:295–308.
31. Suciu LC, Popescu LM, Regalia T, Ardelean A, Manole CG.Epicardium: interstitial Cajal-like cells (ICLC) highlighted byimmunofluorescence. J Cell Mol Med 2009; 13: 771–7.
32. Hinescu ME, Ardeleanu C, Gherghiceanu M, Popescu LM.Interstitial Cajal-like cells in human gallbladder. J Mol Histol2007; 38: 275–84.
33. Popescu LM, Ciontea SM, Cretoiu D. Interstitial Cajal-likecells in human uterus and fallopian tube. Ann N Y Acad Sci2007; 1101: 139–65.
34. Cretoiu D, Ciontea SM, Popescu LM, Ceafalan L, ArdeleanuC. Interstitial Cajal-like cells (ICLC) as steroid hormone sen-sors in human myometrium: immunocytochemical approach. JCell Mol Med 2006; 10: 789–95.
35. Cretoiu SM, Cretoiu D, Suciu L, Popescu LM. Interstitial Cajal-like cells of human Fallopian tube express estrogen and pro-gesterone receptors. J Mol Histol 2009; 40: 387–94.
36. Gherghiceanu M, Hinescu ME, Andrei F, Mandache E, MacarieCE, Faussone-Pellegrini MS, Popescu LM. Interstitial Cajal-like cells (ICLC) in myocardial sleeves of human pulmonaryveins. J Cell Mol Med 2008; 12: 1777–81.
37. Hinescu ME, Popescu LM, Gherghiceanu M, Faussone-Pelle-grini MS. Interstitial Cajal-like cells in rat mesentery: an ultra-structural and immunohistochemical approach. J Cell Mol Med2008; 12: 260–70.
38. Gabella G. Innervation of the gastrointestinal tract. Int RevCytol 1979; 59: 129–93.
39. Cismasiu VB, Radu E, Popescu LM. miR-193 expression dif-¸ ¸ferentiates telocytes from other stromal cells. J Cell Mol Med2011; 15: 1071–4.
40. Herzog EL, Bucala R. Fibrocytes in health and disease. ExpHematol 2010; 38: 548–56.
41. Marchand F. Die ortlichen reaktiven Vorgange (Lehre von derEntzundung). In: Krehl L and Marchand F, editors: Handbuchder allgemeinen pathologie, vol. 4, part 1. Leipzig, 1924; 78.
42. Faussone-Pellegrini MS, Bani D. Relationships between telo-cytes and cardiomyocytes during pre- and post-natal life. J CellMol Med 2010; 14: 1061–3.
43. Faussone-Pellegrini MS. Cytodifferentiation of the interstitialcells of Cajal of mouse colonic circular muscle layer. An E.M.study from fetal to adult life. Acta Anat 1987; 128: 98–109.
44. Faussone-Pellegrini MS, Vannucchi MG, Ledder O, Tian-YingHuang T-Y, Hanani M. Plasticity of interstitial cells of Cajal: astudy of mouse colon. Cell Tissue Res 2006; 325: 211–7.
45. Faussone-Pellegrini MS, Vannucchi MG, Alaggio R, Strojna A,Midrio P. Morphology of the interstitial cells of Cajal of thehuman ileum from foetal to neonatal life. J Cell Mol Med 2007;11: 482–94.
46. Lorincz A, Redelman D, Horvath VJ, Bardsley MR, Chen H,Ordog T. Progenitors of interstitial cells of Cajal in the postnatalmurine stomach. Gastroenterology 2008; 134: 1083–93.
47. Huizinga JD, White EJ. Progenitor cells of interstitial cells ofCajal: on the road to tissue repair. Gastroenterology 2008; 134:1252–4.
48. Gherghiceanu M, Popescu LM. Human epicardium: ultrastruc-tural ancestry of mesothelium and mesenchymal cells. J CellMol Med 2009; 13: 2949–51.
49. Gherghiceanu M, Popescu LM. Cardiomyocyte precursors andtelocytes in epicardial stem cell niche: electron microscopeimages. J Cell Mol Med 2010; 14: 871–7.
Q4:Pleasesupplyvolume andpagenumbers forRefs. 22, 24,53 and 55
Q5:Please checkthe editornames andadd thepublisher’sname forRef. 41
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Maria-Simonetta Faussone-Pellegrini, Professor Emeritusof Histology and Embriologyat the Faculty of Medicine ofthe University of Florence, ismember of the EditorialBoard of the Journal of Cel-lular and Molecular Medi-cine, Assistant Editor of theItalian Journal of Anatomyand Embryology, HonoraryForeign Member of the
Romanian Academy of Medical Science. Her main research-es deal on the interstitial cells of Cajal that in 1977 shesuccessfully proposed to be the intestinal pacemaker cells.She also studies enteric and cerebral neuronal and glial cells,gastrointestinal muscles, stromal cells formerly named inter-stitial Cajal-like cells and now telocytes. All these topicshave been studied by electron microscope and histochemistryin normal and pathological conditions and during differen-tiation. Her publications are about 200, most of which inJournals with IF.
L.M. Popescu, MD, PhD,Dr. h.c.mult., is currentlyProfessor of Cellular andMolecular Medicine, ‘Davi-la’ University of Medicine,Bucharest, Romania andHead of the National Instituteof Pathology, Bucharest,Romania. He is fellow of theNational Academy of Sci-ences and the President ofAcademy of Medical Sci-ences. Recently, he became
President Elect of the Federation of European Academies ofMedicine, and of the International Society for Adaptive Med-icine. He published over 100 scientific articles in interna-tional peer-review journals and is cited more than 1500times. He has a Hirsch Index of about 30. Professor Popescuis Editor-in-Chief (and founder) of the Journal of Cellularand Molecular Medicine (Wiley/Blackwell), with a 5-year IFof 5. He is credited with the discovery of Telocytes.
50. Yamazaki K, Eyden BP. Gap junctions and nerve terminalsamong stromal cells in human fallopian tube ampullary mucosa.J Submicrosc Cytol Pathol 1998: 30: 399–408.
51. Gibbons SJ, De Giorgio R, Faussone-Pellegrini MS, Garrity-Park MM, Miller SM, Schmalz PF, Young-Fadok TM, LarsonDW, Dozois EJ, Camilleri M, Stanghellini V, Szurszewski JH,Farrugia G. Apoptotic cell death of human interstitial cells ofCajal. Neurogastroenterol Motil 2009; 21: 85–93.
52. Mandache E, Gherghiceanu M, Macarie C, Kostin S, PopescuLM. Telocytes in human isolated atrial amyloidosis: ultrastruc-tural remodelling. J Cell Mol Med 2010; 14: 2739–47.
53. Gevaert T, De Vos R, Everaerts W, Libbrecht L, Van Der AaF, van den Oord J, Roskams T, De Ridder D. Characterizationof upper lamina propria interstitial cells in bladders frompatients with neurogenic detrusor overactivity and bladder painsyndrome. J Cell Mol Med 2011; Jan 20. doi: 10.1111/j.1582-4934.2011.01262.x.
54. Gherghiceanu M, Hinescu M, Popescu LM. Myocardial inter-stitial Cajal-like cells (ICLC) in caveolin-1 KO mice. J CellMol Med 2009; 13: 202–6.
55. Cipriani G, Serboiu CS, Gherghiceanu M, Faussone-PellegriniMS, Vannucchi MG. NK-receptors, Substance P, Ano1 expres-sion and ultrastructural features of the muscle coat in Cav-1-/-
mouse ileum. J Cell Mol Med 2011. May 2. doi: 10.1111/j.1582-4934.2011.01333.x.
56. Klumpp D, Horch RE, Kneser U, Beier JP. Engineering skeletalmuscle tissue—new perspectives in vitro and in vivo. J CellMol Med 2010; 14: 2622–9.
57. Polykandriotis E, Popescu LM, Horch RE. Regenerative med-icine: then and now—an update of recent history into futurepossibilities. J Cell Mol Med 2010; 14: 2350–8.
58. Popescu LM, Gherghiceanu M, Kostin S, Faussone-PellegriniMS. Telocytes and heart renewing. In: Wang P, Kuo CH, TakedaN, Singal PK, editors. Adaptation biology and medicine, vol 6.Cell adaptations and challenges. New Delhi: Narosa, 2010:17–39.
59. Gittenberger-de Groot AC, Winter EM, Poelmann R. Epicar-dium-derived cells (EPDCs) in development, cardiac diseaseand repair of ischemia. J Cell Mol Med 2010; 14: 1056–60.
60. Li TS, Cheng K, Lee ST, Matsushita S, Davis D, Malliaras K,Zhang Y, Matsushita N, Smith RR, Marban E. Cardiospheresrecapitulate a niche-like microenvironment rich in stemness andcell-matrix interactions, rationalizing their enhanced functionalpotency for myocardial repair. Stem Cells 2010; 28: 2088–98.
61. Rupp H, Rupp TP, Alter P, Jung N, Pankuweit S, Maisch B.Intrapericardial procedures for cardiac regeneration by stemcells: need for minimal invasive access (Attach Lifter) to thenormal pericardial cavity. Herz 2010; 35: 458–65.
62. Limana F, Capogrossi MC, Germani A. The epicardium in car-diac repair: from the stem cell view. Pharmacol Ther 2011; 129:82–96.
63. Kostin S. Types of cardiomyocyte death and clinical outcomesin patients with heart failure. J Am Coll Cardiol 2011; 57:1532–4.
64. Russell JL, Goetsch SC, Gaiano NR, Hill JA, Olson EN,Schneider JW. A dynamic notch injury response activates epi-cardium and contributes to fibrosis repair. Circ Res 2011; 108:51–9.
65. Popescu LM. The tandem: telocytes—stem cells. Int J Biol Bio-med Eng 2011; 2/5: 83–92.
Received June 28, 2011; accepted August 3, 2011
Page 10 of 16
Table 1. Telocytes distribution.
cavitary organs non-cavitary organs
heart (endo-, myo-, and peri-cardium)
(6,16,19,20,25-29,30,31) lung and pleura (17)
stomach and intestine (12,21) exocrine pancreas (2)
gallbladder (32) mammary gland (1)
uterus (3,33,34) placenta (14)
Fallopian tube (5,35) skeletal muscle (18)
blood vessels (36) mesentery (37)
Page 11 of 16
Figure 1. A and B. CD34-immunoreactivity; submucosa of human stomach. A: the CD34-positive
cells (in brown) have a triangular or ovoid body and a variable number of thin and long prolongations that give a stellate shape to the whole cell. These processes have knobs along their length and a dichotomous branching. Bar: 20 µm. (with permission from ref. 12). B: a detail of the body and prolongations of a CD34-positive cell. Bar = 15 µm. C: human non-pregnant myometrium in cell culture, day 3, the first passage. Giemsa staining. One Telocyte establishing contacts with a smooth muscle cell (myocyte) by a cell process (telopode) of about 65 µm long. Photographic composition of 4 serial phase contrast images; original magnification 40x. In red rectangles, a higher magnification clearly shows the moniliform aspect of the telopode; at least 40 specific
dilations (podoms) of the telopode, interconnected by thin segments (podomeres), are visible in a ‘beadlike’ fashion. Original magnification 40x. (with permission from ref. 3). D: human resting
mammary gland stroma, transmission electron microscopy. One telopode, that appears very long and convoluted, with intercalated podomeres and podoms. Note homocellular junctions marked by
red circles, as well as shed vesicles (SV, blue) and an exosome (violet). coll = collagen. (with permission from ref. 1).
170x108mm (300 x 300 DPI)
Page 12 of 16
Figure 2. A and B: Transmission electron microscope. A: rat mesentery. One telocyte (TC) with a small nucleated body and three long telopodes, all of them having a sinuous trajectory and forming an interstitial complicated 3D network. B: human exocrine pancreas. The telocytes (TC) form with
their typical long telopodes a network around the acini. 180x124mm (300 x 300 DPI)
Page 13 of 16
Figure 3. This electron tomography (thick section of about 300 nm) shows nanostructures connecting one telopode with two adjacent cardiomyocytes in adult mouse heart. The bridging
structures (encircled) have 10-15 nm and suggest a molecular interaction between the telopode and the cardiomyocytes. The dilated segment of this telopode contains a mitochondrion (m). (with
permission from ref. 25). 87x52mm (300 x 300 DPI)
Page 14 of 16
Figure 4. A: human stomach. Double CD34/c-kit labelling. CD34-positivity is in red and c-kit-positivity in green. The CD34- and c-kit-positive cells are often very close to each other but none of
them is double labelled. Bar: 25 µm. B and C. CD34-immunoelectro-labelling. B: mouse small intestine. CD34-positivity is present along the plasma membrane of a long, thin process of a
telocyte (TC), while interstitial cells of Cajal (ICC), nerve fibers and smooth muscle cells (SMC) are CD34-negative. Bar: 1 µm, C: mouse stomach. Detail of a CD34-immunoelectro-labelled telopode.
CD34-positivity clearly appears as electron-dense spherules regularly distributed on the telopode plasma membrane. Bar: 0.4 µm. 180x122mm (300 x 300 DPI)
Page 15 of 16
Figure 5. Schema and electron micrograph illustrating the relationships several telocytes (blue)
have with a column of cardiomyocyte progenitors (CMP, brown) in an epicardial stem cell niche of adult mouse. The telopodes run parallel to the main axis of the CMP column and seem to establish
the direction of development. (with permission from ref. 27). 180x134mm (300 x 300 DPI)
Page 16 of 16
J. Cell. Mol. Med. Vol 14, No 4, 2010 pp. xxx-yyy
Editorial
(Review-Article)
L. M. Popescu a, b,*, Maria-Simonetta Faussone-Pellegrini c, *
a Department of Cellular and Molecular Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romaniab "Victor Babeș" National Institute of Pathology, Bucharest, Romania
c Department of Anatomy, Histology and Forensic Medicine, Section of Histology, University of Florence, Florence, Italy
Accepted: February 25, 2010
Abstract
Ramon y Cajal discovered a particular cell type in the gut, which he named "interstitial neurons" more that 100 years ago. In the
early 1970s, electron microscope (EM) studies showed that indeed a special interstitial cell type corresponding to the cells discov-
ered by Cajal is localized in the gut muscle coat, but it became obvious that they were not neurons. Consequently, they were renamed
"Interstitial Cells of Cajal" (ICC) and considered to be pace-makers for gut motility.
For the past 10 years many groups were interested in whether or not ICC are present outside the gastrointestinal tract, and
indeed, peculiar interstitial cells were found in: upper and lower urinary tracts, blood vessels, pancreas, male and female reproduc-
tive tracts, mammary gland, placenta, and, recently, in the heart as well as in the gut. Such cells, now mostly known as Interstitial
Cajal-Like Cells (ICLC), were given different and confusing names. Moreover, ICLC are only apparently similar to canonical ICC. In
fact, EM and cell cultures revealed very particular features of ICLC, which unequivocally distinguishes them from ICC and all other
interstitial cells: the presence of 2–5 cell body prolongations that are very thin (less than 0.2 mm, under resolving power of light
microscopy), extremely long (tens to hundreds of mm), with a moniliform aspect (many dilations along), as well as caveolae .
Given the unique dimensions of these prolongations (very long and very thin) and to avoid further confusion with other inter-
stitial cell types (e.g. fibroblast, fibrocyte, fibroblast-like cells, mesenchymal cells), we are proposing the term TELOCYTES for them,
and TELOPODES for their prolongations, by using the Greek affix "telos".
Keywords: telocytes • interstitial cells of Cajal (ICC) • interstitial Cajal-like cells (ICLC) • interstitial cells • telopodes •
myocardium • myometrium • mammary gland • genitourinary tract • digestive tract • pancreas • stromal cells •
regenerative medicine
DoI: 10.1111/j.1582-4934.2010.001059.x
TELOCYTES - A case of serendipity:
the winding way from Interstitial Cells of Cajal (ICC),
via Interstitial Cajal-Like Cells (ICLC) to
TELOCYTES
* Correspondence to: L.M. PoPesCu, M.D., Ph.D.Department of Cellular and Molecular Medicine, “Carol Davila” university of Medicine and Pharmacy, P.o. Box 35-29, Bucharest 35, Romania.e-mail: [email protected]
Interstitial Cells of Cajal (ICC)
At first there was…….What was at the beginning? The answer
is: the cells that s. Ramon y Cajal discovered in the muscle
coat of the gut and called “interstitial neurons” more than
100 years ago [1]. He gave them this name since these cells
i) looked like nerve cells, ii) were identifiable through staining
techniques which specifically labelled neurons (e.g. methylene
blue or silver impregnation), and iii) were located in the
interstitium between nerve endings and smooth muscle cells.
However, unbelievably, their “existence” was more or less
rejected by the scientific community of that time. After about
half a century, examination of the gut muscle coat under the
electron microscope revealed cells probably corresponding to
the so-called Cajal’s “interstitial neurons.” Among the pioneers
of this “re-discovery” were M.s. Faussone-Pellegrini [2] and,
independently, L. Thuneberg [3]: it was immediately clear that
these cells were not neurons. Therefore, scientists labelled
them Interstitial Cells of Cajal (acronym: ICC). From then the
“interstitial neurons” of Cajal were buried with all the honours
they deserved and “Interstitial Cells of Cajal (ICC)” were born
and grew (Figs. 1 & 2).
At present, scientific papers (e.g. [4, 5]) are available for:
a) a complete map of the distribution of ICC within the muscle
coat of the gut of several mammals, including humans, b) a
detailed description of their ultrastructural features which is of
great help in their identification, c) molecules expressed by
ICC, considered to be markers for these cells (c-kit) and their
role(s) (NK2r). It has also been demonstrated that ICC origin
is mesenchymal, thus further confirming that they are not
neuronal cells [6].
After the first pioneers, who were morphologists, other
morphologists consider ICC as interstitial cells possibly
forming a functional network, while physiologists see them
as pace-maker cells. All together these researchers agreed
with and confirmed the role proposed for these cells by
Cajal: to be actively involved in the regulation of
gastrointestinal motility, and not only as pace-maker cells
but also in neurotransmission and stretch sensing (see
[7–9] for reviews). Moreover, pathologists view them as the
origin of Gastro-Intestinal Stromal Tumors (GIST) and
being primarily involved in several gastrointestinal
disorders [10]. Apparently, ICC are “great expectations” for
pharmacologists and / or some drug companies, because it
is reasonable to assume that there are millions of cases of
motility disorders of the digestive tract, although there are
no available statistics.
2
Fig. 1 Rabbit colon; circular muscle layer. one interstitial cell of
Cajal (ICC) with an elongated body and three processes located in
the connective interstitium. This cell has close cell-to-cell contact
between both body and processes and two smooth muscle cells
(sMC, arrows) and nerve endings (N, double arrows). This cell has
the typical features of an intramuscular ICC: conspicuous Golgi
apparatus and several rough endoplasmic reticulum cisternae; basal
lamina is thin and discontinous.
1.6 mm
Fig. 2 Human stomach; circular muscle layer. An interstitial cell of
Cajal (ICC) located in the connective interstitium between the cross-
sectioned bundles of smooth muscle cells (sMC), with which ICC
establishes numerous cell-to-cell contacts (asterisks). Note the prox-
imity of a large nerve bundle (N). The ICC body is spindle-shaped and
the cytoplasm is rich in filaments. The basal lamina is thick but dis-
continuous.
4 mm
**
**
**
**
**
**
3
J. Cell. Mol. Med. Vol 14, No 4, 2010
Serendipity ...from ICLC to telocytes
During the last decade, new players appeared in the
ICC field. However, we are faced with a perfect
example of serendipity (see ref. [11]). The term, coined
almost 250 years ago by Horace Walpole, an english
novelist, defines serendipity as the ability of making
fortunate� and� unexpected� discoveries� by� accident
and�sagacity. Louis Pasteur once said, “In the field of
observation, chance only favors the prepared mind.”
There are plenty of instances of serendipity in many
scientific domains, from the discovery of quinine,
vaccines, mast cells, penicillin or X-rays in medical
field, to the detection of uranus in astronomy and even
Teflon®, cellophane and microwaves ....
Let us see what has happened and what might
happen. Fascinated by ICC, Popescu and his team
looked for cells located in pancreas [12, 13] (see
Figs. 3–5), where Cajal also saw his “interstitial
neurons”. The Bucharest team extended their studies
to other organs: myometrium (see Figs. 6 & 7),
fal lopian tube, placenta, mammary gland, gal l
bladder, mesentery, pulmonary veins ([14–22]),
demonstrating the frequency and the ubiquity of the
cell type they found. Noteworthy, a series of
publications was dedicated to the presence and
significance of Interstitial Cajal-like Cells (ICLC) in
mammalian and human myocardium and epicardium
[23–33]; see Figs. 8–10. Many authors also described
cells they were considering more or less ICC and/or
ICLC at least in the upper and lower urinary tracts
([34–38], for reviews see [39]), blood vessels [40–44],
lymphatics [45], as well as male and female
reproductive systems ([46-49], for reviews see [50]).
Popescu et al. performed an eM examination, as well as
staining techniques used by Cajal to label his “interstitial
neurons” and the immunohistochemical methods supposed
to specifically label the ICC, and observed cells with a
typical interstitial location and a phenotype more or less
similar to that of the ICC. The existence of a (“new”) not yet
described cell type became increasingly clear to both: the
Bucharest experts and others. However, the name chosen
was not so (much) different from ICC: interstitial Cajal-like
cells (ICLC) (first used in ref. [15]), since at that time the
aim was to stress how similar these cells were to ICC.
Incidentally, this was not a good idea because, in fact, there
were only a few similarities, not to mention the peculiar
morphology of the ICLC, a morphology that is unique
among all other interstitial cell types.
Fig. 3 Rat exocrine pancreas. Non-conventional light microscopy;
objective 100x. Tissue fixed with glutaraldehyde and postfixed in oso4.
Thin section of epon-embedded material (~ 1 mm) was stained with tolu-
idin blue; cap = capillary; ven = venule. At least 4 telocytes (TC) are pre-
sent in the interstitium among acini (a). Note the cell bodies of TC and
the emerging prolongations - telopodes (dashed lines). The length of the
telopodes is very impressive: tens of mm (!); they are very thin (less than
0.5 mm). Reproduced with permission from [13].
Fig. 4. Immunohistochemistry: Telocytes in human pancreas. Paraffin-
embedded pancreas sections were incubated with polyclonal antibodies
against CD 117. Nuclei were counterstained with Mayer hematoxylin.
Telocytes (arrows) with fusiform body can be seen. having typical long,
moniliform cytoplasmic processes that ‘touch’ the acini. original magnifica-
tion: 100x, oil immersion.
Reproduced with permission from [12].
Fig. 5 Humanexocrine pan-creas.Positive
immunostaining
of telocytes for
CD34 (arrows),
counterstained
with Mayer’s
hematoxylin,
40x.
R e p r o d u c e dwith permissionfrom [13].
TC
TC
TC
TC
4
Fig. 6 Digitally-colored EM image of a telocyte in rat myometrium: telocyte (blue), smooth muscle cells - sMC (sienna-brown); N = Nuclei.
Note three long, moniliform processes that encircle bundles of cross-cut smooth muscle cells. original magnification x 6,800. Inset: Human
pregnant myometrium. Primary confluent culture (day 8) showing a telocyte with at least 3 prolongations with several ‘beads’ along telopodes.
Reproduced with permission from [14] .
Cell cultureElectron microscopy
M
M
Fig. 7 EM of human non-pregnant uterus. Note the telocyte covering
smooth muscle cells (M). The telopode is digitally coloured in blue, marked
with asterisks. Image obtained in 2006.
Courtesy of Prof. M.�Taggart (Newcastle University, UK) and Dr. Carolyn�J.P.�Jones (Manchester University, UK)
T e l oC yT e
T e l oC yT e
t e lo c y t e
( T e l o pod e )
500 nm
5
J. Cell. Mol. Med. Vol 14, No 4, 2010
Telo-
CyTeS
2 mm
TeloCyTe
Telopode
Telopode
Telo
pode
TeloCyTe
Fig. 8. EM image oftelocytes (rat) in theright atrial intersti-tium; telocytes are indi-
cated by arrows. Note
the characteristic
aspects of telopodes:
very long and very thin
cellular elongations,
with uneven calibre
(moniliform aspect).
Rectangles show por-
tions of telocyte body,
containing (abundant)
rough endoplasmic
reticulum (reR).
Reproduced with permis-sion from [30].
Fig. 9. Electron micrograph from cardiac stem cells niche(subepicardium) illustrating the relationships of the telocyte (digital-
ly blue coloured) with cardiomyocyte progenitors - CMP, (brown).
The telocyte processes (telopodes) run parallel with the main axis of
the CMP and seem to establish their direction of development.
Reproduced with permission from [31].
6
Fig. 10. Representative scanning electron micrograph. Monkey left ventricular myocardium. The image shows a typical telocyte locat-
ed across the cardiomyocytes. Another (possible) telocyte appears located near the cardiomyocytes (upper left). The three-dimensional view
reveals close interconnections of ICLCs with cardiomyocytes and capillaries (cap).
Reproduced with permission from [30].
TEloCyTE
telopodetelopode
ICLC continue to be studied with eM and
immunohistochemistry. In particular, in order to best characterize
them and understand their role(s), Popescu’s team tested many
markers. At present, however, only eM gives an unequivocal and
conclusive answer. Immunohistochemistry, on the contrary, has
given answers most of which are confusing because the positivity
to the markers tested is different between organs and animal
species (Fig. 11). Moreover, most of the markers expressed by
the ICLC are in common with several cell types (see CD34 which
labels ICLC and endothelium, c-kit which labels ICC and some
ICLC, etc.). surely ICLC share the same mesenchymal origin with
all these cells, but this origin is a terribly vague marker.
Fig. 11 Human fallopian tube; subconfluent primary culture.
Double immunofluorescent labeling of an ‘octopus’-like telocyte:
vimentin (green) and CD117/ckit (red). Vimentin reactivity is main-
ly localized within the cell processes, and CD117/c-kit has a
patchy pattern. The cell nucleus is shown in blue (Hoechst 33342);
original magnification 60x.
Reproduced with permission from [16].
TeloCyTe
7
J. Cell. Mol. Med. Vol 14, No 4, 2010
Fig 12. Rabbit colon. Two telocytes (asterisks) close to the submucosal border of the circular muscle layer. These cells have a small oval body,
mainly occupied by the nucleus, and extremely thin and long processes extend beyond the cellular body, curving repeatedly. No basal lamina is
present around these cells. sMC = smooth muscle cells.
2 mm
TeloCyTeS
using electron microscopy (Fig. 12), and immuno-
electron microscopy, Faussone-Pellegrini et al. (Fig. 13) re-
examined the muscle coat of the gut. This is a region where
these authors and many other researchers have seen,
described and studied the ICC from all possible angles,
without, however, paying attention to the existence of cells
like ICLC. The results were particularly intriguing: ICLC are
present in the gut muscle coat in great quantities (Fig. 12),
coexist with the ICC and often share an identical
distribution. one of the conclusions of that study was that
ICLC and ICC are two different cell types. Another
collateral conclusion is that we will never know whether the
“interstitial neurons” described by Cajal correspond to the
ICC, to the ICLC or encompass both.
Fig. 13 CD34-immunoelectro-labeling: small intestine. CD34-immunoelectro-labeling is present on the surface of a telocyte. The labelling
appears as an electron-dense material distributed all along the plasma membrane, from which spherules protrude outside.
Reproduced with kind permission from [52].
0.5 mm
telopode
TeloCyTe
8
Topic: Interstitial Cajal-like Cells (now Telocytes)
Fig. 14 The growing scientific interest in “Interstitial Cajal-like Cells” (now TELOCYTES). Charts based on data released by Web of
science (Thomson Reuters ISI Web of Knowledge)
c i t a t i o n sc i t a t i o n sp u b l i s h e d p u b l i s h e d
a r t i c l e sa r t i c l e s
TELOCYTES and TELOPODES
obviously, the cells that we named “ICLC” are different from
ICC. Therefore, it is reasonable (even mandatory) to give them
a different name that refers to them, only. As the “interstitial
neurons” became known as “Interstitial Cells of Cajal”, we think
that the “Interstitial Cajal-Like Cells” (ICLC) should be called
“TeLoCYTes” from now on, by using the Greek affix “Telos”.
Aristotle believed that Telos (Telos) was an object’s or
individual’s greatest potential [11]. The initial meaning of the
word was “burden”, and the most probable semantic
development was from “duty”/“task” to “execution of task”,
“completeness”, and most important, “power to decide” [51].
At present, one could easily see the fast-rate ascending
trend of the interest in “Interstitial Cajal-like Cells” (or ICLC)
during the last 5 years (see Fig. 14). Note the visible difference
between the growing rate of the two parameters (published
items vs. citations), showing a progressive interest in these
cells, which are actually telocytes.
Between 1991 and 2009, a PubMed search of the
Medline database retrieved over 250 records on topic of
Interstitial Cajal-like Cells and more than 7400 citations, with
an average of almost 30 citations per item, consequently
leading to a Hirsch�index of 43.
Telocytes [refs. 12–33]
General aspect of the telocyte is of a small, oval-shaped cellular
body, containing a nucleus, surrounded by a small amount of
cytoplasm. The cellular body average dimensions are, as
measured on eM images, 9.39 mm ± 3.26 mm (min = 6.31 mm;
max = 16.42 mm). The nucleus occupies about 25% of the cell
volume and contains clusters of heterochromatin attached to the
nuclear envelope.
The perinuclear cytoplasm is rich in mitochondria (which occupy
about 5% of the cell body), contains a small Golgi complex, as well
as elements of rough and smooth endoplasmic reticulum and
cytoskeletal elements (thin and intermmediate filaments).
The cell periphery is represented by an usual plasmalemma,
with no (or thin and discontinuous) basal lamina, and many
caveolae (about 2–3% of cytoplasmic volume; ~0.5 caveolae/ mm
of cell membrane length).
The shape of the telocytes is according to the number of their
telopodes: piriform for one prolongation, spindle for two
telopodes, triangular for three, stellate etc. Presumably, their
spatial appearance would be that of a polyhedron with a different
number of vertices, depending on their telopode number.
Telopodes [refs. 12-33, 52]
since we are thinking that telopodes are distinctive for telocytes,
we would like to emphasize at least the following characteristics:
1. number: one to five, frequently only 2–3 telopodes are
observed on a single section, depending on site and angle of
section, since their 3D convolutions impede them to be
observed at their full length in a 2D very thin section;
2. length: tens – up to hundreds of mm, as measured on eM
images (Figs. 6–9, 12, 13). However, under favorable condition
in cell cultures, their entire length can be captured (Fig. 15);
9
J. Cell. Mol. Med. Vol 14, No 4, 2010
T e loCy T e
Fig. 15 Human non-pregnant myometrium in cell culture; day 3; the 1st
passage. Giemsa staining. A telocyte establishing contacts with a myocyte by
a telopode of about 65 mm long. Photographic composition of 4 serial phase
contrast images, original magnification 40x. In red rectangles, a higher mag-
nification clearly shows the moniliform aspect; at least 40 specific dilations are
visible in a ‘bead-like’ fashion.
Reproduced with permission from [14].
3. thickness: uneven caliber, mostly below 0.2 mm (resolving power of
light microscopy), visible under electron microscopy, only 0.10 mm
± 0.05 mm (min = 0.03 mm; max = 0.24 mm; see Figs. 6–9, 12, 13);
4. moniliform aspect, with many dilations along (e.g. Fig. 15);
5. presence of “Ca2+-release units” at the level of the dilations,
accommodating i) mitochondria (as seen by vital staining using
Janus Green B or MitoTraker Green FM, as well as by eM); ii)
elements of endoplasmic reticulum and iii) caveolae;
6. branching, with a dichotomous pattern;
7. organization in a network – forming a labyrinthine system by
tridimensional convolution and overlapping, communicating
through gap junctions.
This characteristic feature makes telopode clearly different from
neuronal dendrites, processes of antigen-presenting dendritic cells or
fibroblasts and myofibroblasts. All the previously mentioned cell
processes (except telopodes) have a thick emergence from the cell
body, followed by gradual thinning.
Noteworthy, except the axons of some type of neurons, telopodes
of telocytes are probably the longest cellular prolongations in the
human body! Furthermore, we have to emphasize that telopodes are
completely different from nerve cells axons or dendrites.
Telocytes have "strategic" positioning in a tissue, in between blood
capillaries and their specific target cells (e.g. smooth muscle cells,
cardiomyocytes) (see Fig. 10 for a typical topography of telopodes) and
in close contact with nerve endings. The distance between telopodes
and myocytes is within the range of tens of nm, which fits the domain of
macromolecular interactions.
Last but not least, to underline that telopodes could establish close
contacts, like synapses, with immunoreactive cells, in various organs,
we called such "connective connections" as stromal synapses [53]
(see Fig. 16).
Why telocytes were not described so far as a distinct cell type ?
Naturally, a question may arise: why so many scientists ignored
telocytes? It is out of question that a cell could be seen under the
microscope, only. Presumably, telocytes were mainly neglected due
to the physical constraints of light and/or electron microscopy
methodology.
a) Light microscopy.The usual stain of H & e could not allow the
differentiation of a telocyte cell body and the cell body of a fibrocyte
/ fibroblast. on the other hand, the area of the microscopic field is
too small to get a clear and complete image of telopodes, which are
anyway below the resolving power (0.2 mm) of light microscopy. In
addition, even the best possible sections obtained using a
microtome (2–4 mm thickness) cannot offer a very good resolution.
In light microscopy, using an oil immersion objective with a
10x eyepiece, the diameter of area of the section is less than
100 mm wide, which will correspond, for instance, to no more
than a row of 4–5 hepatocytes. Therefore, it would be a matter
of luck to catch an entire telocyte with its long and convoluted
telopodes.
b) Electron microscopy uses ultrathin sections of about
60–80 nm. At 2,000x magnification, a regular telocyte
appears too small to be clearly observed. successively
increasing the magnification, in spite of improving the
accuracy of the ultrastructural details, the entire structure fails
to be included in the observation field. A magnification of
about 7,000x shows only a small fragment of the telocyte. At
28,000x magnification, usually, the limited field under
observation does not allow recognition of a telocyte.
Instead of conclusions: why have telocytes?
usualy, people are looking at interstitial cells as being mainly
fibroblasts. However, fibroblasts have the function of generating
connective tissue matrix, specifically collagen. The distinctions
between fibroblasts and telocytes becomes important as their
functions should be mostly different. The expected progress in
knowledge for interstitial cells will show that not all the cells
present in the interstitium, apparently fibroblasts / fibrocytes,
should be labelled as fibroblasts. In other words, other types of
interstitial cells, mainly telocytes, should not be ignored as they
have been in the past.
It may be possible that different locations of telopodes could
be associated with different roles. Anyway, the expression of c-kit
receptors differs between telocyte populations (possible site-
dependent?). Intriguingly, for researchers accustomed with the
pace-maker role of ICC, telocytes presumably exercise other
functions, since they have also been found in non-cavitary
organs, such as pancreas [12, 13], mesentery [21] and even
placenta [20]. Moreover, cardiac telocytes do not function as
pace-maker, but they may influence the rate and rhythm
generated by nodal system.
Hypotethically, many roles were ascribed to telocytes (formerly,
ICLC). However, there is no reasonable evidence to support them.
10
Fig. 17. Electron micrographs show details of telopodesfrom mouse epicardium. shed vesicles (arrowhead) emerge
from telopodes in the interstitial space.
Fig. 16. Rat myometrium: TEM; original magnification 5,700x;image digitally colored. A multicontact synapse (Ms) between an
eosinophil (green) and a telocyte (violet) indicated by cassette. An
unmyelinated nerve bundle (vermilion) appears in proximity of telocyte,
but has no direct contact with it. Apparently, the telocyte ‘prefers’ the
eosinophil
Reproduced with permission from [53].
Fig. 20 electron micrographs show details of telopodes
from mouse epicardium. A. The sinuous and moniliform
telopodes accommodate mitochondria (m), caveolae and eR
cisternae. Calcium release units (arrows) could be seen at
this level. Note close vicinity of telopodes with nerves. B.
shed vesicles (arrow) emerge from telopodes in the intersti-
tial space. C. Gap junction (arrow) between two telopodes.
te lopode
telopode
TeloCyTe
In our opinion telocytes are involved in intercellular signaling,
taking into account the 3D network of telopodes and their strategic
position in between target cells, blood capillary and nerve ending.
At least two mechanisms could be considered: i) a paracrine
and/or juxtacrine secretion of small signal molecules and ii)
shedding microvesicles (see Fig. 17), which play unique roles in
horizontal transfer of important macromolecules among
neighbouring cells (e.g. proteins or RNAs, microRNA included).
such a mechanism, via shed vesicles, may serve to rapid
phenotype adjusment in a variety of conditions [54].
An important role, which could be attributed to telocytes
in the heart is that of being active players in cardiac
renewing, since they are “nursing” cardiomyocyte
progenitors in epicardial stem cell niches [31, 32]. A strong
argument is provided by comparative micro-anatomy: the
newt heart is a model for adult heart regeneration, as newts
can functionally regenerate their heart after amputation of
the apex of the ventricle [55]. In this regenerative process,
a supporting network of stromal cells is primarily developed
and these cells, which fulfill all ultrastructural criteria for
telocytes, are present in large number [56]. Therefore,
apparently an important goal for regenerative medicine
would be to find some factors to stimulate telocytes, as
autologuous in situ cells ...
Epilogue
"There are no small problems. Problems that appear small
are large problems that are not understood”.
Ramon�y�Cajal�[57]
11
J. Cell. Mol. Med. Vol 14, No 4, 2010
References
For�supplementary�materials�on�telocytes�please�see�
www.telocytes.com
1. Ramon y Cajal S. Histologie du systeme
Nerveux de L’Homme et des Vertebres.
Volume 2. Paris: A. Maloine; 1911.
2. Faussone Pellegrini MS, Cortesini C,Romagnoli P. [ultrastructure of the tunica
muscularis of the cardial portion of the
human esophagus and stomach, with spe-
cial reference to the so-called Cajal’s inter-
stitial cells]. Arch Ital Anat Embriol. 1977;
82: 157–77.
3. Thuneberg L. Interstitial cells of Cajal:
intestinal pacemaker cells? Adv AnatEmbryol Cell Biol. 1982; 71: 1–130.
4. Faussone-Pellegrini MS, Thuneberg L.Guide to the identification of interstitial
cells of Cajal. Microsc Res Tech. 1999; 47:
248–66.
5. Faussone-Pellegrini MS. Interstitial cells
of Cajal: once negligible players, now bla-
zing protagonists. Ital J Anat Embryol.2005; 110: 11–31.
6. Lecoin L, Gabella G, Le Douarin N.origin of the c-kit-positive interstitial cells
in the avian bowel. Development. 1996;
122: 725–33.
7. Sanders KM, Koh SD, Ward SM.Interstitial cells of cajal as pacemakers in
the gastrointestinal tract. Annu RevPhysiol. 2006; 68: 307–43.
8. Garcia-Lopez P, Garcia-Marin V,Martinez-Murillo R, Freire M. updating
old ideas and recent advances regarding
the Interstitial Cells of Cajal. Brain ResRev. 2009; 61: 154–69.
9. Huizinga JD, Zarate N, Farrugia G.Physiology, injury, and recovery of intersti-
tial cells of Cajal: basic and clinical scien-
ce. Gastroenterology. 2009; 137:
1548–56.
10. Min KW, Leabu M. Interstitial cells of
Cajal (ICC) and gastrointestinal stromal
tumor (GIsT): facts, speculations, and
myths. J Cell Mol Med. 2006; 10:
995–1013.
11. Misra R. serendipity and science.
Science Reporter. 2008; 45: 9–13.
12. Popescu L, Hinescu M, Radu E, et�al.CD117/c-kit positive interstitial (Cajal-like)
cells in human pancreas. J Cell Mol Med.
2005; 9: 738–9.
13. Popescu LM, Hinescu ME, Ionescu N,et�al. Interstitial cells of Cajal in pancreas.
J Cell Mol Med. 2005; 9: 169–90.
14. Ciontea SM, Radu E, Regalia T, et�al. C-
kit immunopositive interstitial cells (Cajal-
type) in human myometrium. J Cell MolMed. 2005; 9: 407–20.
15. Gherghiceanu M, Popescu LM.Interstitial Cajal-like cells (ICLC) in
human resting mammary gland stroma.
Transmission electron microscope (TeM)
identification. J Cell Mol Med. 2005; 9:
893–910.
16. Popescu LM, Ciontea SM, Cretoiu D, etal. Novel type of interstitial cell (Cajal-
like) in human fallopian tube. J Cell MolMed. 2005; 9: 479–523.
17. Popescu LM, Vidulescu C, Curici A, etal. Imatinib inhibits spontaneous rhythmic
contractions of human uterus and intesti-
ne. Eur J Pharmacol. 2006; 546: 177–81.
18. Hinescu ME, Ardeleanu C,Gherghiceanu M, Popescu LM.Interstitial Cajal-like cells in human gall-
bladder. J Mol Histol. 2007; 38: 275–84.
19. Popescu LM, Ciontea SM, Cretoiu D.Interstitial Cajal-like cells in human uterus
and fallopian tube. Ann NY Acad Sci.2007; 1101: 139–65.
20. Suciu L, Popescu LM, GherghiceanuM. Human placenta: de visu demonstra-
tion of interstitial Cajal-like cells. J CellMol Med. 2007; 11: 590–7.
21. Hinescu ME, Popescu LM,Gherghiceanu M, Faussone-PellegriniMS. Interstitial Cajal-like cells in rat
mesentery: an ultrastructural and immu-
nohistochemical approach. J Cell MolMed. 2008; 12: 260–70.
22. Cretoiu SM, Cretoiu D, Suciu L,Popescu LM. Interstitial Cajal-like cells
of human Fallopian tube express estro-
gen and progesterone receptors. J Mol
12
Histol. 2010; DoI: 10.1007/s10735-009-
9252-z.
23. Hinescu ME, Popescu LM. Interstitial
Cajal-like cells (ICLC) in human atrial
myocardium. J Cell Mol Med. 2005; 9:
972–5.
24. Hinescu ME, Gherghiceanu M,Mandache E, et�al. Interstitial Cajal-like
cells (ICLC) in atrial myocardium: ultra-
structural and immunohistochemical cha-
racterization. J Cell Mol Med. 2006; 10:
243–57.
25. Popescu LM, Gherghiceanu M,Hinescu ME, et�al. Insights into the inter-
stitium of ventricular myocardium: inter-
stitial Cajal-like cells (ICLC). J Cell MolMed. 2006; 10: 429–58.
26. Mandache E, Popescu LM,Gherghiceanu M. Myocardial interstitial
Cajal-like cells (ICLC) and their nano-
structural relationships with intercalated
discs: shed vesicles as intermediates. JCell Mol Med. 2007; 11: 1175–84.
27. Gherghiceanu M, Hinescu ME, AndreiF, et�al. Interstitial Cajal-like cells (ICLC)
in myocardial sleeves of human pulmo-
nary veins. J Cell Mol Med. 2008; 12:
1777–81.
28. Gherghiceanu M, Hinescu ME,Popescu LM. Myocardial interstitial
Cajal-like cells (ICLC) in caveolin-1 Ko
mice. J Cell Mol Med. 2009; 13: 202–6.
29. Gherghiceanu M, Popescu LM. Human
epicardium: ultrastructural ancestry of
mesothelium and mesenchymal cells. JCell Mol Med. 2009; 13: 2949–51.
30. Kostin S, Popescu LM. A distinct type of
cell in myocardium: interstitial Cajal-like
cells (ICLCs). J Cell Mol Med. 2009; 13:
295–308.
31. Popescu LM, Gherghiceanu M, ManoleCG, Faussone-Pellegrini MS. Cardiac
renewing: interstitial Cajal-like cells nurse
cardiomyocyte progenitors in epicardial
stem cell niches. J Cell Mol Med. 2009;
13: 866–86.
32.Gherghiceanu M, Popescu L.Cardiomyocyte precursors and telocytes
in epicardial stem cell niche. electron
microscope images. J Cell Mol Med.
2010; 14.
33.Suciu L, Popescu LM, Regalia T, et�al.epicardium: interstitial Cajal-like cells
(ICLC) highlighted by immunofluorescen-
ce. J Cell Mol Med. 2009; 13: 771–7.
34.Hashitani H, Suzuki H. Identification of
interstitial cells of Cajal in corporal tissues
of the guinea-pig penis. Br J Pharmacol.2004; 141: 199–204.
35.Davidson RA, McCloskey KD.Morphology and localization of interstitial
cells in the guinea pig bladder: structural
relationships with smooth muscle and
neurons. J Urol. 2005; 173: 1385–90.
36.McHale NG, Hollywood MA, SergeantGP, et� al. organization and function of
ICC in the urinary tract. J Physiol. 2006;
576: 689–94.
37.Sergeant GP, Thornbury KD, McHaleNG, Hollywood MA. Interstitial cells of
Cajal in the urethra. J Cell Mol Med. 2006;
10: 280–91.
38.Rasmussen H, Rumessen JJ, HansenA, et�al. ultrastructure of Cajal-like inter-
stitial cells in the human detrusor. CellTissue Res. 2009; 335: 517–27.
39.Hashitani H, Lang RJ. Functions of ICC-
like cells in the urinary tract and male
genital organs. J Cell Mol Med. 2010; DoI:
10.1111/j.1582-4934.2010.01043.x.
40.Bobryshev YV. subset of cells immuno-
positive for neurokinin-1 receptor identi-
fied as arterial interstitial cells of Cajal in
human large arteries. Cell Tissue Res.2005; 321: 45–55.
41.Harhun MI, Pucovsky V, Povstyan OV,et�al. Interstitial cells in the vasculature. JCell Mol Med. 2005; 9: 232–43.
42.Harhun M, Gordienko D, Kryshtal D, etal. Role of intracellular stores in the regu-
lation of rhythmical [Ca2+]i changes in
interstitial cells of Cajal from rabbit portal
vein. Cell Calcium. 2006; 40: 287–98.
43.Pucovsky V, Harhun MI, Povstyan OV,et� al. Close relation of arterial ICC-like
cells to the contractile phenotype of vas-
cular smooth muscle cell. J Cell Mol Med.
2007; 11: 764–75.
44.Morel E, Meyronet D, Thivolet-Bejuy F,Chevalier P. Identification and distribu-
tion of interstitial Cajal cells in human
pulmonary veins. Heart Rhythm. 2008; 5:
1063–7.
45.McCloskey KD, Hollywood MA,Thornbury KD, et�al. Kit-like immunoposi-
tive cells in sheep mesenteric lymphatic
vessels. Cell Tissue Res. 2002; 310:
77–84.
46.Van der Aa F, Roskams T, Blyweert W,De Ridder D. Interstitial cells in the
human prostate: a new therapeutic tar-
get? Prostate. 2003; 56: 250–5.
47.Duquette RA, Shmygol A, Vaillant C, etal. Vimentin-positive, c-kit-negative inter-
stitial cells in human and rat uterus: a role
in pacemaking? Biol Reprod. 2005; 72:
276–83.
48.Shafik A, Shafik I, el-Sibai O.Identification of c-kit-positive cells in the
human prostate: the interstitial cells of
Cajal. Arch Androl. 2005; 51: 345–51.
49.Dixon RE, Hwang SJ, Hennig GW, et�al.Chlamydia infection causes loss of pace-
maker cells and inhibits oocyte transport
in the mouse oviduct. Biol Reprod. 2009;
80: 665–73.
50.Hutchings G, Williams O, Cretoiu D,Ciontea SM. Myometrial interstitial cells
and the coordination of myometrial
contractility. J Cell Mol Med. 2009; 13:
4268–82.
51.Waanders F. Constructions and mea-
nings of Teloς. The history of TEΛOΣ
and TEΛEΣ in ancient Greek.
Amsterdam: B.R. Grüner Publishing Co.,
1983. pp. 231–7.
52.Pieri L, Vannucchi MG, Faussone-Pellegrini MS. Histochemical and ultra-
structural characteristics of an interstitial
cell type different from ICC and resident in
the muscle coat of human gut. J Cell MolMed. 2008; 12: 1944–55.
53.Popescu LM, Gherghiceanu M, CretoiuD, Radu E. The connective connection:
interstitial cells of Cajal (ICC) and ICC-like
cells establish synapses with immuno-
reactive cells. electron microscope study
in situ. J Cell Mol Med. 2005; 9: 714–30.
54.Cocucci E, Racchetti G, Meldolesi J.shedding microvesicles: artefacts no
more. Trends Cell Biol. 2009; 19: 43–51.
55.Borchardt T, Kostin S. Plastizitat von
Herzmusklezellen des Molches. Max-Plack-Gesellschaft, Entwicklungs-&Evolutionsbiologie. 2007; 237–43.
56.Popescu L, Gherghiceanu M, Kostin S,Faussone-Pellegrini M. Cardiac
Telocytes (Interstitial Cajal-Like Cells) and
Heart Renewing. In: Wang P, Kuo C,
Takeda N, singal P, eds. Adaptation
Biology and Medicine: Narosa Publishers,
2010 (in press).
57.Ramon y Cajal S. Advice for a young
investigator. The MIT Press; Cambridge,
MA; 1999.
