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Stem cells (2)

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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Stem cells (2)

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 3 Stemcells(2)

  3. Cord blood stem cells • Approx. 130 millionbabiesbornyearly – theumbilicalcordblood is thelargestpotentialsource of stemcellsforregenerativemedicine • Inthepast 36 yrs 10000 patientsweretreatedfor over 80 differentdiseases

  4. Cord blood stem cells and fetal stem cells Cord blood collection from umbilical vein(after birth) 1 2 3 Add cord blood Express Express Analysis of blood Cell separation The cord blood cells are frozen in bag or cryovials Mix and settle Concentrate cells Freeze cells 35 min 10 min Liquid nitrogen storage tank(-150°)

  5. 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.

  6. Cord blood processing • Red cell depletion (using Ficoll, Hetastarch, Lymphoprep, Prepacyte) • Depletion of plasma for smaller storage size • Testing of the final cell pool (infection, volume, cellularity, stem cell content, CD34+)

  7. Cord bloodprocessing and cryopreservation • Cord blood is primarily useful in hematological disorders • Cord blood is collected at birth • Either processed or just simply frozen in DMSO

  8. Cord bloodbanking • Cord blood banks should be set up inevery metropolitan city with HLY specification and linked to an international computer network • Keeping cord blood for a considerable length of time is costly

  9. Pluripotenciy of cordbloodstem cells Cord Blood Purification Stem cells CBE MSC Endodermal Mesodermal Ectodermal Hepato-Biliary Blood Neural

  10. Stemcellpopulationincordblood Adherent MSC Pre-MSC CBE Non-adherent Lin- CD133+ CD34+ CordBlood Bonemarrow PeripheralBlood

  11. Disorders treatable with cord bloodI Oncologic disorders Immune deficiencies 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 • 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

  12. Disorders treatable with cord bloodII Hematological disorders 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 Metachromaticleukodystrophy Mucolipidosis Types II, III Neimann Pick syndrome, Types A and B Sandoff syndrome Sanfilippo syndrome Tay Sachs disease • 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

  13. Fat stem cells(ASC) • Fat or adipose tissue stem cells (ASC): • Easily obtainable • Consistent immunophenotype • Similar to BMSC • Multipotent • Manipulation by genetic engineering

  14. Types of adipose tissues

  15. Isolation procedures Wash in PBS 300g, 5 min Aspiration of lipocytes Digestion withcollagenase at 37oC, 1hr Stromalvascular fraction (SVF)

  16. Immunophenotype of ASCsPositive markers

  17. Immunophenotype of ASCsNegative markers

  18. Cytokine profile of ASCs

  19. 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.

  20. Differentiation potential of ASCs • Adipocyte • Cardiac myocytes • Chondrocyte • Endodermal and ectodermal lineages • Endothelial and smooth muscle cells • Hematopoietic support • Neuronal lineage • Osteoblast • Skeletal myocytes

  21. Differentiation into adipocytes

  22. Differentiation into cardiacmyocytes

  23. Differentiation into chondrocytes

  24. Differentiation into osteocytes

  25. Differentiation into skeletalmyocytes

  26. Differentiationintoneuronalcells

  27. Differentiationintoendodermal and ectodermallineages

  28. Differentiationintoendothelial and smoothmusclecells

  29. Hematopoieticsupport

  30. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 4 Stemcells(3)

  31. Application of ESCs and ASCs Embryonic development Endoderm Ectoderm Blastocyst Inner cell mass ESCs Lung Brain Neuron Astrocyte Oligodendrocyte NSCs Subventricular zone niche Hippocampus niche (dentate gyrus region) Pluripotent ESC BASCs Bronchioalveolar duct junction niche Endodermal stem cell Ectodermal stem cell Mesodermal stem cell Lung disorders Brain and spinal cord disorders Hemangioblast Eye • Pancreas • Insulin-secreting b-cells RSCs Ciliaryepithelium niche Mesoderm Heart Cardiomyocytes Exocrine acini Cornea Endocrine islets of Langerhans CESCs Limbus niche CSCs Atria niche Apex niche Retina PSCs Pancreatic duct putative niche Eye disorders Skin Diabetes Cardiac diseases Liver Hepatocytes KSCs Basal layer niche Bone EPC HSC MSC bESCs eNCSCs Bulge niche HSCs Endosteum surface niche Microvasculature niche Osteoblasts Chondrocytes Adipocytes Myoblasts Common myeloid precursor Common lymphoid precursor HDCs Bile duct (canal of Hering) MSCs Perivascular surface niche SKPs Dermal papilla NK-cells Platelets T-lymphocytes Erythrocytes Hepatic disorders Skin disorders B-lymphocytes Granulocytes Hemopoietic and Immune system disorders Dendritic cells Monocytes Macrophages Gene therapies Bloodstream Neutrophil Vascular lumen NK-cell Intravenous injection Genetically modified stem cell-based delivery HSC EPC Macrophage Platelets Eosinophil T-lymphocyte Erythrocyte Basophil B-lymphocyte Monocyte New endothelial cell Dendritic cell Media Vascular wall-resident stem cells EPCsandMSCs Vascular disorders Vasculogenic zone

  32. Genetic engineering and gene delivery using ASCs • Lentiviral vectors can transduce ASCs • Other recombinant viral vectors • Nucleofection

  33. Approaches and methods for controlling stem cell growth and differentiation Transduction with lineage specific genes can help todrive differentiation. Reporter tags like GFP aid selection (i.e. FACS) and reveal when and where genes are activated Biochem factors added to culture medium (including serum) stimulate differentiation. Requires knowledge of factors likely to induce differentiation but is rarely, if ever, 100% effective. Gene Reporter (e.g. GFP) Bioreactors come in many 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 to grow larger pieces of tissue. Cell sorting techniques like MACS or FACS can positively select (or negatively deplete unwanted cell types) using cell surface antibodies or fluorescent transduced markers like GFP. FACS MACS Co-culture with the cells or tissues 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 Differential adhesion assays using specific ECM proteins or receptor ligands can helpinencouragingselection of specific cell types based on affinity and kinetics of cell-substratum interactions. Related to this is the colony forming unit (CFU) assay. Scaffold can provide 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.

  34. Reprogramming Virus carries reprogramming factors intosomatic cell’s nucleus Pluripotent iPSC line Somatic cell is reprogrammed Culture as perhESCs

  35. Differentiation of Cells I Precursor cell Regulatory protein 1 Cell division Regulatory protein 2 Regulatory protein 2 Regulatory protein 3 Regulatory protein 3 Regulatory protein 3 Regulatory protein 3 Cell C Cell D Cell G Cell H Cell A Cell B Cell E Cell F

  36. Differentiation of Cells II Germ cells Sperm Egg Gastrula Ectoderm (External layer) Blastocyst Zygote Skin cells of epidermis Neuron of brain Pigment cell Mesoderm (Middle layer) Cardiac muscle Skeletal muscle cells Red blood cells Smooth muscle Tubule cell of the kidney Endoderm (Internal layer) Lung cell (Alveolar cell) Thyroid cell Pancreatic cell

  37. Mature, organ specific primary cellsI Cell culture Biopsy Purification Cells for engineering

  38. Mature, organ specific primary cellsII Purification Cells for engineering Tissue specific resident stem cell Biopsy Cell cultures Differentiated tissue cells

  39. Differentiation of epidermis Cornifiedenvelope Lipidenvelope Extrusion of lipidsfromlamellargranules Filaggrin, loricin, trichohyalin, involucrin, SPRRs, S100 proteins Stratumcorneum Granular layer Keratin-2e Epidermaldifferentiation Spinous layer Keratin-1, -10 Basal layer Keratin-5, -14 Basal membrane

  40. Mature tissue specific cellsin tissue engineering • Biopsy or resection • Purification • Regaining proliferation capacity in cell culture • Re-differentiation

  41. Regulatory issues ICells • GLP • GMP • Permit to work on ES

  42. Regulatory issues IIAnimals • Permission to work on animals • UK: Home Office Licence 1986 • EC 1394/2007

  43. RegulatoryissuesIIIHuman EmbryonicStemCells • Ethicalissues of using human embryosassources of stemcells

  44. Regenerative medicine • Organ regeneration by inducing self-regenerative biochemical and cellular processes • Organ regeneration by addition of in vitro generated full organs or specific tissues of an organ

  45. 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

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