basic principles
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Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
BASIC PRINCIPLES
Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 1
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Aim of the courseTo provide theoretical background knowledge to complex tissue cultures
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Course contentsWeek Lecture Title
1 1 Basic principles
2 Stem cells (1)
2 3 Stem cells (2)
4 Stem cells (3)
3 5 Bioreactors (1)
6 Bioreactors (2)
4 7 Biomaterials (1)
8 Biomaterials (2)
5 9 Scaffold fabrication
10 Biocompatibility
6 11 Cell-Scaffold interaction
12 Biofactors
7 13 Controlled release
14 Biosensors
Week Lecture Title
8 15 Aggregation cultures
16 Tissue printing
9 17 Tissue repair (1)
18 Tissue repair (2)
10 19 Tissue repair (3)
20 Tissue repair (4)
11 21 Commercial products (1)
22 Commercial products (2)
12 23 Clinical trials (1)
24 Clinical trials (2)
13 25 Clinical trials (3)
26Research applications and drug testing
14 27 Ethical Issues
28 Economic significance
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What is tissue engineering?• Tissue engineering was previously known as a
subfield of engineering and biomaterials, but having grown in scale and significance tissue engineering has become a discipline of its own.
• Tissue engineering aims to regenerate or recreate specific tissues or full organs with maintained or recreated biological function (bone, cartilage, blood vessels, bladder, skin, etc).
• To achieve the aims tissue engineering uses a combination of techniques including cell culture, engineering bio-materials, biochemical and biophysical methods.
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Basic principles of tissue engineering
Monolayer cell cultureExpanded cell culture
Generation of a graft
Cells from a biopsy or resection
Grafting Biopsy
Culture in 3D conditions
(scaffold based or scaffold free)
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2D tissue culturesAdherent Non-Adherent
Suspension
Pelleting
Re-suspension
Seeding
Trypsin No trypsin
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3D tissue cultures
No diffusion
Apoptotic death
Secondary necrosis
Necrotic tissue
Diffusion of nutrients and oxygen
Healthy tissue
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Engineering tissuesIn vitro
• Tissue regeneration (tissue proliferation and regeneration on artificial scaffold)
• Bio-artificial organs (liver, skin, pancreas)
In vivo• Blood cell regeneration
(stimulation of stem cell proliferation and differentiation)
• Cell injection into injured tissues (myocardiocytes)
• Large injury regeneration (bio-absorbable scaffolds with or without growth factors)
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Bioreactor• Bioreactors are needed to avoid necrosis
Spinner flask bioreactor
Peristaltic pumps
Drive
Water in
Water out
Air
Counterpressurevalve
Electromagneticvalvefor cooling
Pump
Safety valve
Process Contoller
Heater vessel
Acid BaseAntifoam Substrate
Q
Q valve
Foam
T
pH
pO2
Industrial bioreactor
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Potential uses of cell and tissue replacement therapy• Stroke• Traumatic brain injury• Alzheimer’s disease• Parkinson’s disease• Missing teeth• Wound healing• Bone marrow
transplantation• Spinal cord injury• Osteoarthritis• Rheumatoid arthritis
• Crohn’s disease• Baldness• Blindness• Deafness• Myocardial infarction• Muscular dystrophy• Diabetes• Multisite cancers• Amyotrophic lateral
sclerosis
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Cells in tissue engineeringStem cells• Embryonic• Cord blood• Bone marrow or adipoid tissue derived• Adult, tissue specific Mature differentiated cells• Tissue specific
STEM CELLS (1)
Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 2
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Stem cell types
Neural cells
Blood cells
Cultured stem cells
8 cell embryo
Fertilizedoocyte
Totipotent
Blastocyst
Pluripotent
Multipotent
Brain Neural cells Blood cellsBone marrow
Multipotent
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Types of stem cell replications I
Asymmetric, replicating,
differentiating division
Symmetric, differentiating
division
Symmetric, replicating
division
Parental stem cell
Daughter cell Self-reneweddaughter cell
Self-renewal
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Types of stem cell replications IIProliferation capacity
Differentiation capacity
Term
inal
ly d
iffer
entia
ted
cells
Stem cell
Proliferating compartmen
t
Asymmetric divisionAmplification of cell
numbersDifferentiation via progenitor cells
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Sources of Stem Cells• Adult (somatic) stem cells (ASC)• Adult multipotent stem cells• Embryonic stem cells
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Epiblast stem cells (EpiSC) IEpiblast is a tissue that forms at a later stage than ES cells. The epiblasts forms after the developing embryo implants into the uterus.
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Epiblast stem cells (EpiSC) II
Primordialgerm cells (GSC)
Fertilization
Morula
Embryonicstem cells (ESC)
Epiblast
Late blastocyst
EpiSCs
Somatic cell lineages• Endoderm• Mesoderm• Ectoderm
IPS cells
Dedifferentiationand reprogramming
Embryonicgerm cells (EG)
Extraembryonicendoderm (XEN) cells
Primitiveendoderm
Trophoblaststem cells (TS)
Trophectoderm(TE)
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Location of multipotent adult somatic stem cells was identifiedFormerly identified in:
Recently stem cells have been identified
in practically all tissues
BrainSkin
Bone marrow
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Adult (somatic) stem cells (ASC)The microenvironment where stem cells live are called stem cell
niches:Stem cells from fat
Peripheral blood Bone marrow
Skeletal muscle Placenta Hair follicle
Cartilage Bone marrow Cartilage Skeletal muscle Bone marrow SkinMuscle Nerve Tendon Smooth muscle Nerve BrainBone Blood cells Muscle Heart Cartilage Smooth muscleNerve Fat Bone Tendon Fat
Liver Fat Blood vesselsBone marrow CartilageBrain/nerve
BloodHeart
All tissues
ASC in:
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The adult stem cell nicheStem cell niches defined as microenvironments:
Gastrointestinal Brain Bone marrowEsophagus Brain CorneaSmall intestine Nerves RetinaLarge intestine/colon Blood cells PancreasStomach Muscle Liver
All tissues HeartLungSpermatogoniaAmniotic fluidUmbilical cord matrixCord bloodVarious tissues
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Marrow stem cells (MSCs)• Hematopoietic stem cells• Mesenchymal stem cells• Bone Marrow Stromal Stem cells• Endothelial Progenitor cells
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Hematopoietic stem cells (HSC) IThyloLin-Sca-1+
• CD4 and Mac-1 based fractionation resolved three populations and only the Lin-Mac-1-CD4- population was highly enriched for long-term reconstituting HSCs
• c-kit+Sca-1+ThyloFlk-2- long-term repopulating HSC and c-kit+Sca-1+ThyloFlk-2+ short-term repopulating HSC
• Long term repopulating HSCs in mice c-kit+ThyloLin-(Flk-2-) Sca-1+
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Hematopoietic stem cells (HSC) II• In human samples CD34 antigen (function
unknown) is expressed• Additional markers: CD38, CDE90 and CD133• Thy+CD34+ long-term cultures generating
both myeloid and lymphoid lineages• Thy-CD34+ no long-term cultures generating
both myeloid and lymphoid lineages
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Prolymphocyte
Monocyte
Hematopoietic stem cells (HSC) III
Hematopoiesis in Humans Multipotential hematopoietic stem cell(Hemocytoblast)
Myeloblast
Common myeloidprogenitor
Common lymphoidprogenitor
Megakaryoblast
Promegakaryoblast
Megakaryocyte
Thrombocytes
Proerythroblast(Pronormblast)
Basophilic erythroblast
Polychromatic erythroblast
Orthochromatic erythroblast (Normoblast)
Polychromatic erythrocyte (Reticulocyte)
Erythrocyte
Mast cell Lymphoid dendritic cell
Myeloid dendritic cell
Plasma cellMacrophage
Small lymphocyteNatural
killer cell
T lymphocyte B lymphocyte
Lymphoblast
B. promyelocyte
B. myelocyte
B. metamyelocyte
B. band
Basophil
N. promyelocyte
N. myelocyte
N. metamyelocyte
N. band
Neutrophil
E. promyelocyte
E. myelocyte
E. metamyelocyte
E. band
Eosinophil
Monoblast
Promonocyte
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Mesenchymal stem cells (MSC) I• Lacking hematopoietic markers (CD2, CD3,
CD4, CD8, Mac-1/CD11b, CD14, CD15, CD19, CD20, B220, CD45, Thy1 and myeloperoxidase)
• Express: LDL receptor, alkaline phosphatase, smooth muscle actin, type IV collage, laminin, factor VII, MUC18, CD29, CD44, CD49A-F, CD51, CD73, CD105, CD106, CD166+
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Mesenchymal stem cells (MSC) II• MSCs can directly be isolated from bone
marrow based on NGFR (CD271), SSEA-1, SSEA-4, CD140b , CD340 (HER-2), CD349 (Frizzled9)
• Purification is based on: CD29+, CD44+, CD73+, CD105+, CD106+, CD166+ and lack of hematopoietic receptors
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Endothelial progenitor cells (EPC)• Adult EPCs CD34+ or Flk-1+ (VEFR-2)• Additional markers: CD31, Tie2 and E-
selectin, eNOS, LDLR, VEGFR-1,-2
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Bone marrow progenitor cells• HSC and EPC lineages follow similar temporal
and spatial development• HSC and EPC shared markers: VEGFR-2 (Flk-
1), SCL/Tal1, Runx1• Existence of the hemangioblast
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Pericytes SMCs
ECs
Ontogeny of tissue lineages in bone marrow
HSCs Osteoblasts
EPCsMSCs
Stromal Cells
Lymphoid progenitor
s
Myeloid progenitor
sMonocyte
s
T-lineage B-lineage
Meso-AngioblastHemangioblast(Flk-
1+VEcadherin+CD45-)
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