b. wozniewicz md phd

NEW TRENDS IN PARATHYROID ALLOTRANSPLANTATION,

Warsaw 2006

B. Wozniewicz MD PhD

Department of PathologyThe Childrens’ Memorial Health Institute, Warsaw

Biomedical Research Center, Warsaw

New trends in parathyroid allotransplantation - 2001

LyMac

End Fib

End

PTH

PTH PTH PTHPure PTH

Cell Culture TX

PTH

PTH

Ly

Ly

PTH

PTHPTH

PTH

PTH

PTH

PTH

PTH

No HLA Class II

PTH TX


Organ PTx Cell PTx

1. Devoid of immunogens2. Controlled selection3. No Rejection4. No immunosupression

1. Immunogenic epitopes 50%2. Surgical blind selection3. Rejection reaction4. Immunosupresion

PTH + HLA class I and II type antigen presentation before and after culturing

Human cultured parathyroid cells injected into a mouse peritoneal cavity confirmed the lack ofimmunogenicity

(one year after xenotransplantation)

Microencapsulated cell cultureAnalysis of cell number and

distribution

Preparates of beadcross section

formaldehyde fixingparaffin – microtomehematoxylin, eosin

stainingNumber of viable cells

Cell culture conditionsCulture period – three weeksChang medium + human serumHumidified atmosphere 370C

5% CO2

Fixed samples of microencapsulated cell culture

Photos made after cell death

Alginate beads transferredto 0.9%NaCl + EtOH Olympus optical microscope, transmitted light

thanks to dr Korzynska, IBIB

New trends in parathyroid allotransplantation – 2001

Non Rejection Reaction

• 1. Problem of adhesion-failure• 2. Problem with capillary finding• 3. Problem with vasculature interaction• 4. Problem with programmed death• 5. Problem with local ischaemia• 6. Problem with monitoring • 7. Problem with recovery of HLA class-I


Supported by

Grant Nr PB 538/PO5/98/15State Committee for Scientific Research

Participants:

Prof.dr. T.Tołłoczko, Prof. dr . J. Szmit & Dr med. I. Nawrot Medical School Warsaw

Prof. Dr med. A. Gorski –Institute of Transplantology, Medical School Warsaw

Prof. Dr med. J.Kawiak & Prof. Dr Eng. A. WerynskiIBiIB Warsaw

Dr n med A. Sawicki Calcium Metabolism Center, Osteomed Warsaw

Prof. Dr hab. B.WozniewiczDep.Pathology, CMHI -Warsaw

Bogdan Woźniewicz

The Children’s Memorial Health Institute

Woźniewicz B*, Czarnowska E*, Cukrowska B*, Szperl M**, Leszek P**, Korewicki J**, Religa G**, Religa Z**, Kołsut P**, Grajkowska W*

* The Children’s Memorial Health Institute **Institute of Cardiology , Warsaw,Poland

Introduction. In human and experimental animals there is now incontrovertible evidence that new myocytes are continuously generated throughout life in response to physiological and pathological stimuli. Locally or systematically derived mesodermal cells can differentiate into a variety of mesodermal tissues including bone, cartilage, tendon, fat, endothelium, skeletal muscles and cardiomyocytes. Adult heart stromal cell system has been proposed to consist of mesodermal stem cells that are capable of self-renewal and differentiation into de novo cardiomyocytes. Angiogenesis, gene therapy and cell transplantation are among the newest treatments to improve the still grim outcome of patients with cardiac failure.

Purpose. Topographic sections from explants of patient who underwent heart transplantation were investigated in term of cardiogenesis in the heart with heart failure with stage IV NYHAMethods. Histological, immunocytochemical and electron microscopical investigations were performed on 10 human heart explants with antibodies anti - CD3, CD4, CD34, CD31, PCNA, Ki67, TGF, heavy chain alpha myosin, actin, connexin, cadhedrin, and TUNEL. Two-month culture of heart fragments using Chang medium +20% Fetal Calf Serum was performed.Results. Proliferated cells that represent myofibroblasts and cardiomyoblasts differentiation have been shown at the periphery of scars, in the endocardial layer and in the perivascular space. These findings may be used in prospective studies in selection of these cells for further differentiation and as a reservoir of the cells for cell transplantation therapy of heart failure in future. Cell population resembling phenotypes of myofibroblast–like cells grown slowly in vitro from the fragments obtained from patient with heart failure.

Conclusion. Failing hearts in contrast to normal exhibit accumulation of number of cells being bipotential in differentiation into fibroblast and myofibroblast de novo. In vitro efforts should be made with using a blocking agents, e.g. anti-fibroblastic growth factors to inhibit fibrillogenesis and improve cardiomyogenesis.

Actin Actinin Myosin Vimentin Desmin GATA-4* *Connexin *Catenin NKx2.5Case 1 +++ +++ + ++ + - - - *Case 2 ++ ++ + + + * * * *Case 3 ++ ++ + ++ + * * * *Case 4 + * * * *Case 5 * * * *Case 6 - - - -Case 7 - - - - ++ - - - *Case 8 - - - - +Case 9 ++ ++ ++ + + * * * *

Case 10 ++ ++ ++ + -Control - - - + N+ N+ N+

„Cardiomyogenesis”: origin of the cells

Perivascular myocytes or pericytes transformation

Endothelial cells transformation Resting stromal cells (bipotential) fibromyoblasts.Mesenchymal cardiohistiocytes (Anitchkow cells)Bone marrow cellsPeripheral blood circulating reserve cellsFibroblasts

Fig. 1a

Fig. 1dFig. 1c

Fig. 1b Fig. 2a Fig. 2b

Fig. 2c Fig. 2d

Fig. 3 Fig. 5

Fig. 7

Fig. 4

Fig. 8Fig. 6 Table. 1 Table. 2

Fig.1. Histology of heart explant with IV NYHA post infarct failure. H&E staining. Aggregates of small cells in the periphery of the scar (1b) , in subendocardial 1ayer (1a,c) and perivascular space (1d) are evident suggesting self-renewal fibromyoid cell proliferation. Not seen in normal heart! Mag. 200x. Fig.2. Immunostainig for alpha heavy chain myosin exhibits strong positive reaction in fibromyoid cells (Fig.2 a,b,d. Control reaction in persistent hypertrophic giant cardiomyocytes (2c). The scar with fibroblast is negative (2a). Fig. 3,4,5. Electron microscopic features of fibromyoblastic cells taken from selected area visualised in Fig 1a. The cytoplasm is pourly organised. Intercellular space is devoid of collagen, (3,4), the fibromyoid cells contain basement membrane like material (arrow) and in cytoplasm prominent myofilaments (arrow). OsO4fixation. Mag. Fig 3.4. 20.000x . Fig.5 -40.000x. Fig.6. In contrast to fibromyoblastic cells fibroblasts from the scar exhibit strong potential to collagen production. OsO4 fixation. Mag.20.000x. Fig.7, 8. A population of the cells obtained by culture of the fragments of the heart explants with severe heart failure. During prolonged culture (within 2 month) growth of cells imitated the features to those visualised above on H&E staining (Fig.7), while fragments of normal hearts did not grown in the same conditions. These cells were characterised by positive expression of vimentin (Fig.8). Table. 1. Immunocytochemical features of cardiomyoblast/ fibromyoblasts. Table .2 . Hypothetical origin of cardiomyoblast in severe heart failure.

toronto poster.ppt

Wozniewicz B**, Czarnowska E**, Cukrowska B**, Szperl M*, Leszek P*, ReligaG*, Kołsut P*, Grajkowska W**, Szymańska-Dębińska T**, Korewicki J*, Religa

Z*.*Institute of Cardiology , Warsaw PL **Children’s Memorial Health

Institute, Warsaw PL

Introduction. In human and experimental animals there is now incontrovertible evidence that new myocytes and angiocytes may be generated throughout life in response to physiological and pathological stimuli. Adult heart stromal cell system has been proposed to consist of mesodermal stem cells that are capable of self-renewal into de novo cardiomyocytes. Similar stimuli e.g. prolonged hypoxy is able to stimulate de novo angiogenesis from endothelial cells. The rate of cell renewal is slow and low. In contrast to normal healthy heart the more severe heart failure the more chance in findings of angiogenesis and cardiogenesis is possible. The problem is haw to differentiate natural vascular system from neo-endotheliogenesis and vasculogenesis could be determined. This findings may be enhancement by application of various method of direct or indirect stimulation e.g. plasmides or autologous blood stem cell transplantation.

Aim. The aim of presentation is to introduce electron microscopic investigation for evaluation normal vessels from self-renewal angiocytes. Material and method. Electron microscopic examination were performed in specimens obtained from seven heart explains in patient with stage IV heart insufficiency (NYHA) undergoing heart transplantation.Material was performed in the routine way to Epon 812 (fixation in 3% glutaraldehyde, dehydration, OsO4 staining ). Semithin section stained with toluidine blue were selected using optic microscopy and ultrathin section were observed in Jeol 100CX EM. Additional staining using monoclonal CD31/PACAM-1 and polyclonal VEGF-1 antibodies were used for visualisation of vessels in paraffin embedded sections.Results. Monoclonal and polyclonal antibodies stain adult opened terminal vessels filled usually with erythrocytes. not aggregates of immature cells. Electron microscopy was useful to determine endothelial/angiocytes precursors from other cells since they present basement membrane and fenestration of the cytoplasm. Histologically in the routine examination angiogenesis was suggested when around the cardiocytes were observed not a single vessel but a plexus of vessels and endothelial cells.

Criteria of de novo vasculogenesis

Marker VEGF-1

CD31/Pecam

Immature cell aggregates - -Maturated cells with openings + +

Immunocytochemical

Adult capillaries + + StructureImmature cellaggregates

BM present

Maturated cellsFenestration presentCytopl. “openings” present

Electron microscopic

Adultcapillaries

Lumen +BM+++Membrana fenestrata +

Table. 1. Ultrastructural and immunocytochemical evidence of neovasculogenesis in heart explands with heart failure.

Fig. 1. Anti CD31/PECAM-1 monoclonal antibodies staining of capillaries in normal heart . Magn. 200x.

Fig. 2. Polyclonal antibodies anti VEGF-1 in heart explant with IV NYFA heart failure. De novo capillaries were stained positive. (arrow).Mag. 220x

Fig. 3. Electron microscopy of immature cells exchibiting presence of basement membrane like structure. OsO4 staining. Magnification 20.000 X

Fig. 4. Electron microscopy of two immature endothelial cells with opening (lumen) formation. OsO4 staining.Magnification 20.000X

Fig. 5. Immature angiocytes not forming de novo vessels becameapoptotic (arrow). OsO4 staining.Magnification 20.000X

Conclusion. Electron microscopy is useful in qualification of self-renewal angiogenesis in the heart with severe failure.

IV Konferencja Otolaryngologów Polskich i Francuskich

Warszawa 18-19 Listopada 2005

HODOWLA KOMHODOWLA KOMÓÓREK REK PROGENITOROWYCH PROGENITOROWYCH Z Z

PRZEWODU PRZEWODU ŚŚLIMAKOWEGO LIMAKOWEGO

CultureCulture ofof progenitorprogenitor cellscells fromfrom thethe

cochleacochleaB. Cukrowska1, K. Niemczyk2, K. Pietrasik2, A. Bruzgielewicz2, K. Morawski2,

B. Biskup2, M. Pronicki1, A. Zajączkowska1, B. Woźniewicz1

1 Instytut „Pomnik - Centrum Zdrowia Dziecka” Zakład Patologii Kierownik Doc. dr hab. M. Pronicki

2 Katedra i Klinika Otolaryngologii Akademii Medycznej w WarszawieKierownik Prof. dr hab. n med. K. Niemczyk

Thymic progenitor cells

Narodowy program regeneracji i transplantacji Grasicy 2007-2009

Transplantacje komórek• Realizacja u człowieka• Przytarczyce od 1993 Warszawa skuteczne• Trzustka wiele ośrodków nieskuteczne• Komórki nerwowe dopamina zwierzęta • Komórki sercowe – w toku• Komórki akustyczne experymenty, zwierzeta• Komórki grasicy – USA skutecznie• Komórki wątrobowe – experymenty zwierzeta• Skóra – skuteczne• Keratinocyty - skuteczne

b. wozniewicz md phd

Documents