Transcript Stem cells
Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Three dimensional tissue cultures and tissue engineering – Lecture 3 STEM CELLS (2) TÁMOP-4.1.2-08/1/A-2009-0011 Cord blood stem cells • Approx. 130 million babies born yearly – the umbilical cord blood is the largest potential source of stem cells for regenerative medicine • In the past 36 yrs 10000 patients were treated for over 80 different diseases TÁMOP-4.1.2-08/1/A-2009-0011 Cord blood stem cells and fetal stem cells Cord blood collection from umbilical vein (after birth) 1 2 3 Add cord blood Express Express The cord blood cells are frozen in bag or cryovials Analysis of blood Cell separation Mix and settleConcentrate cells Freeze cells 35 min Liquid nitrogen storage tank (-150°) 10 min TÁMOP-4.1.2-08/1/A-2009-0011 Cryopreservation • Cryopreservation of primary cells is possible for long term (so far 20 yrs). • The low-temperature is maintained at 150-196oC in liquid nitrogen. TÁMOP-4.1.2-08/1/A-2009-0011 Cord blood processing 1. Red cell depletion (using Ficoll, Hetastarch, Lymphoprep, Prepacyte) 2. Depletion of plasma for smaller storage size 3. Testing of the final cell pool (infection, volume, cellularity, stem cell content, CD34+) TÁMOP-4.1.2-08/1/A-2009-0011 Cord blood processing and cryopreservation • Cord blood is primarily useful in hematological disorders • Cord blood is collected at birth • Either processed or just simply frozen in DMSO TÁMOP-4.1.2-08/1/A-2009-0011 Cord blood banking • Cord blood banks should be set up in every metropolitan city with HLY specification and linked to an international computer network • Keeping cord blood for a considerable length of time is costly TÁMOP-4.1.2-08/1/A-2009-0011 Pluripotenciy of cord blood stem cells Cord Blood Purification Stem CBEcellsMSC Endodermal Mesodermal HepatoBiliary Blood Ectoderma l Neura l TÁMOP-4.1.2-08/1/A-2009-0011 Stem cell population in cord blood Adherent MSC Pre-MSC CBE Non-adherent Lin- CD133+ CD34+ Cord Blood Bone marrow Peripheral Blood TÁMOP-4.1.2-08/1/A-2009-0011 Disorders treatable with cord blood I Oncologic disorders Immune deficiencies Acute lymphoblastic leukemia Acute myeloid leukemia Autoimmune lymphoproliferative disorders Burkitt lymphoma Chronic myeloid leukemia Cytopenia related to monosomy Familial hystocytosis Hodgkin’s disease Juvenile myelomonocytic leukemia Langerhans cell hystocytosis Myelodysplastic syndromes Non-Hodgkin’s lymphoma Ataxia telangiectasia Cartilage-hair hypoplasia Chronic granulomatous disease DiGeorge syndrome Hypogammaglobulinaemia IKK gamma deficiency Immune dysregulation polyendocrinophaty Mucolipidosis type II Myelokathesis Severe combined immunodeficiency Wiscott-Aldrich syndrome X-linked agammaglobulinaemia, immunodeficiency, lymphoproliferative syndrome TÁMOP-4.1.2-08/1/A-2009-0011 Disorders treatable with cord blood II Hematological disorders Autoimmune neutropenia Cyclic neutropenia Diamond Blackfran anemia Evan’s syndrome Red cell aplasia Refractory anemia Severe aplastic anemia Sickle cell disease Thalassaemia Fanconi’s anemia Galnzmann’s disease Congenital sideroblastic anemia Juvenile dermatomyositis and xanthogranulomas Metabolic disorders Adrenoleukodystrophy Alpha mannosidosis Type I diabetes Gaucher’s disease Gunther disease Hermansky-Pudlak syndrome Hurler syndrome Hurler-Scheie syndrome Krabbe’s disease Maroteau-lamy syndrome Metachromatic leukodystrophy Mucolipidosis Types II, III Neimann Pick syndrome, Types A and B Sandoff syndrome Sanfilippo syndrome Tay Sachs disease TÁMOP-4.1.2-08/1/A-2009-0011 Fat stem cells (ASC) Fat or adipose tissue stem cells (ASC): • • • • • Easily obtainable Consistent immunophenotype Similar to BMSC Multipotent Manipulation by genetic engineering TÁMOP-4.1.2-08/1/A-2009-0011 Types of adipose tissues TYPE FUNCTION 1 Bone marrow Fills in space no longer used for hematopoiesis 2 Brown adipose tissue (BAT) Protects vital organs in the newborn 3 Ectopic adipose tissue Abnormal fat accumulation in the liver, skeletal or cardiac muscle (e.g. in metabolic syndrome) 4 Mammary adipose tissue Lactation – energy and nutrient source 5 Mechanical adipose tissue Protection from mechanical trauma 6 White adipose tissue (WAT) Insulation, energy storage, reservoir, endocrine organ TÁMOP-4.1.2-08/1/A-2009-0011 Isolation procedures Wash in PBS Aspiration of lipocytes 300g, 5 min Digestion with collagenase at 37oC, 1hr Stromal vascular fraction (SVF) TÁMOP-4.1.2-08/1/A-2009-0011 Immunophenotype of ASCs Positive markers Marker Name Marker Name CD9 Tetraspan CD55 Decay accelerating factor CD10 Common acute lymphocytic leukemia antigen CD59 Protectin CD13 Aminopeptidase CD71 Transferrin CD29 b1-integrin CD73 5’-ectonucleotidase CD34 Sialomucin CD90 Thy1 CD44 Hyaluronate receptor CD105 Endoglin CD49d a4-integrin CD146 Muc-18 CD54 Intracellular adhesion CD166 molecule Activated leukocyte cell adhesion molecule HLA-ABC Histocompatibility locus antigen-ABC a-smooth muscle actin a-SMA TÁMOP-4.1.2-08/1/A-2009-0011 Immunophenotype of ASCs Negative markers Marker Name Marker Name CD11b ab-integrin CD50 Intracellular adhesion molecule-3 CD14 LPS receptor CD56 Neural cell adhesion molecule CD16 Fc receptor CD62 E-selection CD18 b2-integrin CD104 B4-integrin CD45 Common leukocyte antigen HLA-DR Histocompatibility locus antigen-DR TÁMOP-4.1.2-08/1/A-2009-0011 Cytokine profile of ASCs CYOTOKINE Adiponectin Leptin • Plasminogen activator inhibitor-1 • • FUNCTION GROUP Adipokines • • • Hepatocyte growth factor Pigment epithelial derived factor Vascular endothelial growth factor Angiogenic • • • • • Flt-3 ligand GCSF Leukemia inhibitory factor IL-7 MCSF Hematopoietic • • • IL-6, IL-8, IL-11 Leukemia inhibitory factor TNFa Pro-inflammatory TÁMOP-4.1.2-08/1/A-2009-0011 Immunogenecity of ASCs • Lack of immunogenicity is linked to the absence of the major histocompatibility class II antigens (HLA-DR) on their surface. • Their immunosuppressive properties are linked to prostaglandin E2 production. TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation potential of ASCs • • • • • • • • • Adipocyte Cardiac myocytes Chondrocyte Endodermal and ectodermal lineages Endothelial and smooth muscle cells Hematopoietic support Neuronal lineage Osteoblast Skeletal myocytes TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation into adipocytes ORIGIN OF ACS WAT etc. INDUCING AGENT MARKERS INDUCED APPLICATION Forskolin (AMP agonist) Neutral lipids Functional fat pads Methylisobutylxanthine (AMP agonist) Adiponectin Plastic surgery Glucocorticoid receptor ligands (dexamethasone) CAAT/enhancer binding protein-a fatty acid binding protein (aP2) Cosmetic and reconstructive surgery PPAR-g2 ligands (thiazolidinediones) Leptin Insulin Lipoprotein lipase bFGF PPAR-g2 TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation into cardiac myocytes ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED BAT 5-azacytadine Sarcomeric actinin WAT Cardiomyocyte extract Connexin-43 Desmin troponin-I APPLICATION Repair injured cardiac tissue after ischemic injury TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation into chondrocytes ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED Bone marrow adipose tissue Ascorbate Aggrecan BAT Dexamethasone Chondroitin sulfate WAT TGF-b Collagen type II 3D structure Collagen type IV BMP-6 Chondrocyte specific proteoglycans FGF APPLICATION Knee, hip chondroid tissue TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation into osteocytes ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION WAT Ascorbate Osteocalcin Bone implantation Bone marrow adipose tissue Dexamethasone DMP-1 Bone fracture repair 1,25-dihydroxy vitamin D3 Osteoadherin B-glycerophosphate BMP-2 BMP-7 Runx2 TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation into skeletal myocytes ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED Low concentration FBS myoD Horse serum myogenin Myosin light chain kinase APPLICATION TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation into neuronal cells ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED Indomethacin Glial fibrillary acidic protein (GFAP) Insulin Nestin methylisobutylxanthine Intermediate filament Glutamate receptor subunits S-100 B-III tubulin APPLICATION Central nervous system injury Differentiation into endodermal and ectodermal lineages TÁMOP-4.1.2-08/1/A-2009-0011 ORIGIN OF ACS Bone marrow adipoid tissue INDUCING AGENT MARKERS INDUCED Hepatocyte growth factor Albumin Oncostatin M A-fetoprotein DMSO together with HGF, bFGF, nicotinamide Urea ATRA Cytokeratin-18 APPLICATION Liver Epithelial tissue repair (Crohn’s disease) Differentiation into endothelial and smooth muscle cells TÁMOP-4.1.2-08/1/A-2009-0011 ORIGIN OF ACS WAT INDUCING AGENT MARKERS INDUCED APPLICATION CD31 Vascular trauma calponin Urogenital trauma A-smooth muscle actin TÁMOP-4.1.2-08/1/A-2009-0011 Hematopoietic support ORIGIN OF ACS INDUCING AGENT ASCs secrete: IL6,IL7, IL8,IL11 SCF, TNFa, MCSF, GMCSF MARKERS INDUCED CD34+ into T, NK, B markers APPLICATION For patients requireng hematopoietic stem cells reconstruction following high-dose chemotherapy Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Three dimensional tissue cultures and tissue engineering – Lecture 4 STEM CELLS (3) TÁMOP-4.1.2-08/1/A-2009-0011 Application of ESCs and ASCs Embryonic development Endoderm Blastocyst Lung Ectoderm Inner cell mass ESCs Brain Neuron NSCs Subventricular zone niche Hippocampus niche (dentate gyrus region) Pluripotent ESC BASCs Bronchioalveolar duct junction niche Endodermal Mesodermal stem cell Ectodermal stem cell stem cell Lung disorders Brain and spinal cord disorders Hemangioblast Eye Pancreas Insulin-secreting b-cells Exocrine acini Astrocyte Oligodendrocyte Endocrine islets of Langerhans Heart Cornea Cardiomyocytes CESCs CSCs Atria niche Apex niche PSCs Pancreatic duct putative niche Diabetes Retina Limbus niche Eye disorders Skin Cardiac diseases Liver Bone Hepatocytes HDCs Bile duct (canal of Hering) Hepatic disorders RSCs Ciliary epithelium niche Mesoderm EPC HSCs Endosteum surface niche Microvasculature niche HSC MSC Common myeloid Common lymphoid precursor precursor Osteoblasts Chondrocytes Adipocytes Myoblasts MSCs Perivascular surface niche NK-cells Platelets Erythrocytes T-lymphocytes Granulocytes B-lymphocytes Dendritic cells Monocytes Hemopoietic and Immune system disorders Macrophages HSC Platelets Erythrocyte NK-cell Bloodstream Neutrophil Vascular lumen EPC Eosinophil Macrophage Basophil New endothelial cell T-lymphocyte B-lymphocyte Monocyte Dendritic cell Media Vasculogenic zone Vascular disordersVascular wall-resident stem cells EPCs and MSCs KSCs Basal layer niche bESCs eNCSCs Bulge niche SKPs Dermal papilla Skin disorders Gene therapies Intravenous injection Genetically modified stem cell-based delivery TÁMOP-4.1.2-08/1/A-2009-0011 Genetic engineering and gene delivery using ASCs • Lentiviral vectors can transduce ASCs • Other recombinant viral vectors • Nucleofection Approaches and methods for controlling stem cell growth and differentiation TÁMOP-4.1.2-08/1/A-2009-0011 Transduction with lineage specific genes can help to drive differentiation. Reporter tags like GFP aid selection (i.e. FACS) and reveal when and where genes are activated Gene Reporter (e.g. GFP) Cell sorting techniques like MACS or FACS can positively select (or negatively deplete unwanted cell types) FACS MACS using cell surface antibodies or fluorescent transduced markers like GFP. Differential adhesion assays using specific ECM proteins or receptor ligands can help in encouraging selection of specific cell types based on affinity and kinetics of cell-substratum interactions. Related to this is the colony forming Scaffold can provide unit (CFU) assay. physical (e.g. surface roughness, porosity, etc.) and also biochemical (e.g. controlled release of doped growth factors) cues to promote attachment, recruitment, differentiation and delivery of cells. Biochem factors added to culture medium (including serum) stimulate differentiation. Requires knowledge of factors likely to induce differentiation but is rarely, if ever, 100% Bioreactors come in many effective. sizes and designs and include stirred, rotary and perfused systems. All serve to improve exchange efficiency of nutrients and waste products and delivery of growth factors to enable longer term culture, helping to scale-up cell numbers or Co-culture with thepieces cells or to grow larger of tissues tissue. of interest (i.e. the target for tissue repair) can help to encourage differentiation. This can include direct physical contact and/or indirect biochemical signaling TÁMOP-4.1.2-08/1/A-2009-0011 Reprogramming Virus carries reprogramming factors into somatic cell’s nucleus Somatic cell is reprogrammed Pluripotent iPSC line Culture as per hESCs TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation of Cells I Precursor cell Regulatory protein 1 Cell division Regulatory protein 2 Regulatory protein 3 Cell A Cell B Cell C Regulatory protein 2 Regulatory protein 3 Cell D Cell E Regulatory protein 3 Cell F Cell G Regulatory protein 3 Cell H TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation of Cells II Germ cells Gastrula Sperm Egg Ectoderm (External layer) Blastocyst Zygote Skin cells ofNeuron of brain Pigment cell epidermis Mesoderm (Middle layer) Cardiac muscle Skeletal muscle Red blood cellsSmooth muscle Tubule cell cells of the kidney Endoderm (Internal layer) Lung cell Thyroid cell Pancreatic cell (Alveolar cell) TÁMOP-4.1.2-08/1/A-2009-0011 Mature, organ specific primary cells I Cell culture Biopsy Purification Cells for engineering TÁMOP-4.1.2-08/1/A-2009-0011 Mature, organ specific primary cells II Purification Biopsy Tissue specific resident stem cell Differentiated tissue cells Cell cultures Cells for engineering TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation of epidermis Cornified envelope Lipid envelope Epidermal differentiation Extrusion of lipids from lamellar granules Stratum corneum Granular layer Filaggrin, loricin, trichohyalin, involucrin, SPRRs, S100 proteins Keratin-2e Spinous layer Keratin-1, -10 Basal layer Keratin-5, -14 Basal membrane TÁMOP-4.1.2-08/1/A-2009-0011 Mature tissue specific cells in tissue engineering • Biopsy or resection • Purification • Regaining proliferation capacity in cell culture • Re-differentiation TÁMOP-4.1.2-08/1/A-2009-0011 Regulatory issues I Cells • GLP • GMP • Permit to work on ES TÁMOP-4.1.2-08/1/A-2009-0011 Regulatory issues II Animals • Permission to work on animals • UK: Home Office Licence 1986 • EC 1394/2007 TÁMOP-4.1.2-08/1/A-2009-0011 Regulatory issues III Human Embryonic Stem Cells Ethical issues of using human embryos as sources of stem cells TÁMOP-4.1.2-08/1/A-2009-0011 Regenerative medicine • Organ regeneration by inducing selfregenerative biochemical and cellular processes • Organ regeneration by addition of in vitro generated full organs or specific tissues of an organ TÁMOP-4.1.2-08/1/A-2009-0011 Organ failure • Organ failure due to disease, accident or aging requires full organ replacement or regeneration • Ideally, one’s own tissues (autologue) should provide the necessary biomaterial for generation of such organs