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Stem cells


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Stem cell therapy, Basics , History of First stem cell product.

Published in: Health & Medicine
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Stem cells

  1. 1. Stem Cells SUDARSAN AGARWAL Pinnamineni medical college, vijayawada
  2. 2. Definition of Stem Cells • A cell that has the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues • Biological cells found in all multicellular organisms. • Blank or Unspecialized and Undifferentiated cells.
  3. 3. What can be done with Stem Cells? • Have immune potential and can help to treat a wide range of Medical problems. • Discovery of stem cells lead to a whole new branch of medicine known as ….. Regenerative medicine
  4. 4. Historical perspectives • 1908: The term "stem cell" was coined by Alexander Maksimov • 1963: McCulloch and Till illustrate the presence of self-renewing cells in mouse bone marrow. • 1968: Bone marrow transplant between two siblings successfully treats SCID. • 1978: Haematopoietic stem cells are discovered in human cord blood. • 2000: Several reports of adult stem cell plasticity are published. • 2007: The Nobel Prize was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells". • 2008: Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts • 2012: The Nobel Prize was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka "for the discovery that mature cells can be reprogrammed to become pluripotent"
  5. 5. Mouse embryonic stem cells with fluorescent marker
  6. 6. Human embryonic stem cell colony on mouse embryonic fibroblast feeder layer
  7. 7. Human embryonic stem cells Cell colonies that are not yet differentiated Nerve cell
  8. 8. Properties of Stem Cells • Self.renewal.Can regenerate into only the specific tissue from which they are isolated (Proliferation and renewal) • Potency (ES cells) : Capacity to differentiate into specialized cell types of stem cells.
  9. 9. Properties of Stem Cells • Totipotent – Can differentiate into embryonic and extra embryonic cell types. Can construct a complete viable organism. Ex: Cell produced by fusion of an egg and sperm • Pluripotent – Can differentiate into nearly all cells, cells derived from any of the three germ layers. • Multipotent : Can differentiate into a number of cells, but only those of a closely related family of cells. • Oligopotent : Can differentiate into only a few cells, such as lymphoid or myeloid stem cells. • Unipotent : Can produce only one cell type of their own, property of self renewal.
  10. 10. Stem cell division and Differentiation 1: Symmetric stem cell division 2: Asymmetric stem cell division 3: Progenitor division 4: Terminal differentiation A: Stem Cell B: Progenitor C: Differentiated Cell Cell
  11. 11. Types of Cultured Stem Cells • Embryonic stem cells • Embryonic germ cells • Trophoblast stem cells • Extra embryonic endoderm cells • Embryonic carcinoma cells • Mesenchymal stem cells • Epistem cells • Multipotent adult stem cells • Spermatogonial stem cells • Germ line stem cells • Neural stem cells • Unrestricted somatic stem cells • Multipotent adult germ line stem cells • Induced pluripotent stem cells
  12. 12. Embryonic Stem Cells • Derived from inner cell mass of a blastocyst or earlier morula stage. • Blastocyst is an early stage embryo. • 4 – 5 days old – 50 – 150 cells. • „ES‟ cells – Pluripotent – gives rise all three primary germ layers. • Do not contribute to extra embryonic membranes or the placenta.
  13. 13. Adult Stem Cells • It is undifferentiated cells found among differentiated cells in a tissue or organ after birth. • Do not disappear following birth. • Remain, play a role in the recovery of damaged tissue. • Decreased reserve and loss of vitality as with age. • Adult stem cells can also develop into other types of cells and are mostly recovered by bone marrow. • The use of Adult stem cell in research is not controversial as the use of „ES‟ cell, as it does not require the destruction of an embryo.
  14. 14. Adult Stem cells Normal differentiation • Hematopoietic stem cells – give rise to all types of blood cells. • Epithelial stem cells – in GIT – Absorptive cells, goblet cells, entero endocrine cells. • Skin stem cells. Adult Stem Cells Plasticity/ Trans Differentiation • Ability to differentiate into multiple cell types is called plasticity. • Haemopoietic stem cells – differentiate into Brain cells, neuron, oligodendrocytes, skelet al muscle cells, cardiac muscle cells, liver cells etc., • Bone Marrow Stem cells – into cardiac, skeletal or muscle cells. • Brain stem cells – into Blood cells and skeletal muscle cells.
  16. 16. Nuclear reprogramming • Development naturally progresses from totipotent fertilized eggs to pluripotent epiblast cells, to multipotent cells, and finally to terminally differentiated cells. • According to Waddington's epigenetic landscape, this is analogous to a ball moving down a slope. • The reversal of the terminally differentiated cells to totipotent or pluripotent cells (called nuclear reprogramming) can thus be seen as an uphill gradient that never occurs in normal conditions.
  17. 17. Nuclear reprogramming • However, nuclear reprogramming has been achieved using nuclear transplantation, or nuclear transfer (NT), procedures (often called "cloning"), where the nucleus of a differentiated cell is transferred into an enucleated oocyte
  18. 18. DOLLY (5 July 1996 – 14 February 2003)
  19. 19. Dolly Sex Born Died Female 5 July 1996 (Roslin Institute) 14 February 2003 (aged 6) Nation United Kingdom Known for First mammal to be cloned from an adult somatic cell Offspring 6 lambs Named after Dolly Patron
  20. 20. Keith Campbell Dolly Ian Wilmut
  21. 21. • Dolly the sheep, first mammal to be cloned from an adult somatic cell • Even though Dolly was not the first animal to be cloned, she gained this attention in the media because she was the first to be cloned from an adult cell
  22. 22. Somatic Cell Nuclear Transfer
  23. 23. • In Dolly, nucleus was transferred came from mammary gland cells from a 6-year-old ewe • In Polly & molly fibroblast cells were used
  24. 24. Species cloned • • • • Tadpole: (1952) Carp: (1963) Mice: (1986) Sheep: first mammal being cloned (1984) from early embryonic cells. Megan and Morag from differentiated embryonic cells (1995) Dolly from a somatic cell (1996) • Rhesus Monkey: (2001) • Cattle: (2001) • Rat: • Mule: • Horse: • Dog: • Wolf: • Water Buffalo: • Pyrenean Ibex (2009) was the first "extinct" animal cloned • Camel: (2009) • Pashmina goat: (2012) Kashmir , India
  25. 25. Human cloning
  26. 26. Human cloning • Human cloning is the creation of a genetically identical copy of an existing or previously existing human. • The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction.
  27. 27. • Therapeutic cloning involves cloning adult cells for use in medicine and is an active area of research. • Reproductive cloning would involve making cloned humans. A third type of cloning called replacement cloning is a theoretical possibility, and would be a combination of therapeutic and reproductive cloning. • Replacement cloning would entail the replacement of an extensively damaged, failed, or failing body through cloning followed by whole or partial brain transplant
  28. 28. Induced Pluripotent stem cells
  29. 29. Induced Pluripotent Stem Cells • Not adult stem cells. • Reprogrammed cells (Ex. Epithelial cells) will have pluripotent capabilities. • Using genetic reprogramming with protein transcription factors, pluripotent stem cells equivalent to ES cells have been derived from human adult skin tissue. • Little is known about factors that induce this reprogramming. • Induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions.
  30. 30. Nobel prize in physiology or medicine 2012 for the discovery that “mature cells can be reprogrammed to become pluripotent" John B Gurdon Shinya Yamanaka
  31. 31. Reprogramming cells
  32. 32. Induced Pluripotent Stem Cells
  33. 33. Stem Cells APPLICATIONS
  34. 34. Tissue Repair Regenerate spinal cord, heart tissue or any other major tissue in the body.
  35. 35. Heart Disease Adult bone marrow stem cells injected into the hearts are believed to improve cardiac function in victims of heart failure or heart attack
  36. 36. Leukemia and Cancer • Studies show leukemia patients treated with stem cells emerge free of disease. • Injections of stem cells have also reduces pancreatic cancers in some patients. Proliferation of white cells
  37. 37. Rheumatoid Arthritis Adult Stem Cells may be helpful in jumpstarting repair of eroded cartilage.
  38. 38. Parkinson's disease Major motor features of disorder results from the loss of a single cell population i.e., dopaminergic neurons with in stratum nigra. This suggests that cell replacement should be adequate to treat it.
  39. 39. Type I Diabetes • Pancreatic cells do not produce insulin • Embryonic Stems Cells might be trained to become pancreatic islets cells needed to secrete insulin.
  40. 40. Diseases that are treated by stem cells are: 1) Acute Leukemia • Acute Lymphoblast Leukemia (ALL) • Acute Myelogenous Leukemia (AML) • Acute Biphenotypic Leukemia • Acute Undifferentiated Leukemia 2) Chronic Leukemia • Chronic Myelogenous Leukemia (CML) • Chronic Lymphocytic Leukemia (CLL) • Juvenile Chronic Myelogenous Leukemia (JCML) • Juvenile Myelomonocytic Leukemia (JMML) Syndromes • Myelodysplastic Syndromes • Amyloidosis • Chronic Myelomonocytic Leukemia (CMML) • Refractory Anemia (RA) • Refractory Anemia with Excess Blasts (RAEB) • Refractory Anemia with Excess Blasts in Transformation • (RAEB-T) • Refractory Anemia with Ringed Sideroblasts (RARS)
  41. 41. Disorders 1) Stem Cell Disorders • Aplastic Anemia (Severe) • Fanconi Anemia • Paroxysmal Nocturnal Hemoglobinuria • Congenital Cytopenia • Dyskeratosis Congenita 2) Myeloproliferative Disorders • Acute Myelofibrosis • Agnogenic Myeloid Metaplasia • Polycythemia Vera • Essential Thrombocythemia 3) Lymphoproliferative Disorders • Non-Hodgkin’s Lymphoma • Hodgkin’s disease • Prolymphocytic Leukemia 4) Phagocyte Disorders • Chediak-Higashi Syndrome • Chronic Granulomatous Disease • Neutrophil Actin Deficiency • Reticular Dysgenesis 5) Inherited Metabolic Disorders • Mucopolysaccharidoses (MPS) • Hurler’s Syndrome (MPS-IH) • Scheie Syndrome (MPS-IS) • Hunter’s Syndrome (MPS-II) • Sanfilippo Syndrome (MPS-III) • Morquio Syndrome (MPS-IV) • Maroteaux-Lamy Syndrome (MPS-VI) • Sly Syndrome, Beta-Glucuronidase Deficiency • Adrenoleukodystrophy • Mucolipidosis II (I-cell Disease) • Krabbe Disease • Gaucher’s Disease • Niemann-Pick Disease • Wolman Disease • Metachromatic Leukodystrophy 6) Histiocytic Disorders • Familial Erythrophagocytic Lymphohistiocytosis • Histiocytosis-X • Hemophagocytosis • Langerhans’ Cell Histiocytosis
  42. 42. 7) Inherited Immune System Disorders • Ataxia-Telangiectasia • Kostmann Syndrome • Leukocyte Adhesion Deficiency • DiGeorge Syndrome • Bare Lymphocyte Syndrome • Omenn’s Syndrome • Severe Combined Immunodeficiency • SCID with Adenosine Deaminase Deficiency • Absence of T & B Cells SCID • Absence of T Cells, Normal B Cell SCID • Common Variable Immunodeficiency • Wiskott-Aldrich Syndrome • X-Linked Lymphoproliferative Disorder Other Inherited Disorders • Lesch-Nyhan Syndrome • Cartilage-Hair Hypoplasia • Glanzmann Thrombasthenia • Osteopetrosis • Adrenoleukodystrophy • Ceroid Lipofuscinosis • Congenital Erythropoietic Porphyria • Sandhoff Disease 9) Plasma Cell Disorders • Multiple Myeloma • Plasma Cell Leukemia • Waldenstrom’s Macroglobulinemia • Amyloidosis Abnormalities 1) Inherited Platelet Abnormalities Congenital Thrombocytopenia 2) Inherited Erythrocyte Abnormalities • Beta Thalassemia Major • Sickle Cell Disease • Blackfan-Diamond Anemia • Pure Red Cell Aplasia Other Malignancies • Ewing Sarcoma • Neuroblastoma • Renal Cell Carcinoma • Retinoblastoma • Brain tumor • Ovarian Cancer • Small Cell Lung Cancer • Testicular Cancer
  43. 43. Strategies for transplantation of stem cells 1. Undifferentiated or partially differentiated stem cells may be injected directly in the target organ or intravenously.(HSC) 2. Stem cells may be differentiated ex vivo prior to injection into the target organ. (Beta cells, Cardiomyocyte) 3. Growth factors or other drugs may be injected to stimulate endogenous stem cell populations(EPO, GM-CSF)
  45. 45. Bone marrow transplantation was the original term used to describe the collection and transplantation of hematopoietic stem cells, but with the demonstration that the peripheral blood and umbilical cord blood are also useful sources of stem cells, hematopoietic cell transplantation has become the preferred generic term for this process.
  46. 46. Properties of Hematopoietic stem cells • Dies • Self renewal • Potency • Plasticity -- Apoptosis -- Stem cell -- Differentiated blood cells(Differentiation) -- Neurons/ Germ cells( Trans Differentiation)
  47. 47. The procedure is usually carried out for one of two purposes: (1) to replace an abnormal but nonmalignant lymphohematopoietic system with one from a normal donor, or (2) to treat malignancy by allowing the administration of higher doses of myelo suppressive therapy than would otherwise be possible The time it takes for hematopoietic progenitors to become mature cells is ~10–14 days in humans,
  48. 48. In humans, transplantation replaces recipient's entire lymphohematopoietic system, including 1. all red cells, 2. granulocytes, 3. B and T lymphocytes, 4. platelets, 5. Kupffer cells of the liver, 6. pulmonary alveolar macrophages, 7. osteoclasts, 8. Langerhans cells of the skin, and 9. brain microglial cells.
  49. 49. Pre transplant • Whole Body Irradiation to remove endogenous immune system and tumor cells • Injection of bone marrow from a well matched donor to re-establish immune system • Regulation of immune response to prevent graft versus host reaction. • Autologous donation possible if one can purify and remove tumor cells, enriching for stem cells.. • Allogeneic donors have advantage of graft versus tumor reaction to kill any remaining tumor cells. • Allogeneic donors have the disadvantage of graft versus host reaction if they are not well matched.
  50. 50. Purify from Tumor cells Unfractionated Bone marrow Transplant Transplant Donor blood formation, graft-vstumor effect
  51. 51. Complications of Allogeneic Transplants • Regimen related toxicity • Infectious complications • Engraftment failure (resistance) • Graft-versus-host disease • Transplant related mortality = 10 - 15%
  52. 52. Bone Marrow / Ph Blood • Found in spongy bone where blood cells form • Bone marrow aspirated from the posterior and anterior iliac crests has traditionally been the source of hematopoietic stem cells for transplantation. Typically, anywhere from 1.5 to 5 x 108 nucleated marrow cells per kilogram • Used to replace damaged or destroyed bone marrow with healthy bone marrow stem cells. • Hematopoietic stem cells circulate in the peripheral blood but in very low concentrations. • Following the administration of certain hematopoietic growth factors, (EPO,GCSF,GM-CSF) , the concentration of hematopoietic progenitor cells in blood increases markedly.
  53. 53. Umbilical cord Blood Stem Cells • Also Known as Wharton‟s Jelly • Adult stem cells of infant origin • Less invasive than bone marrow • Greater compatibility • Less expensive • Obtained from cord immediately after birth. • Rich source of haemopoietic stem cells • These are referred to as neonatal stem cells, less mature than those found in adults bone marrow.. Three important functions: 1. Plasticity: Potential to change into other cell types like nerve cells 2. Homing: To travel to the site of tissue damage 3. Engraftment: To unite with other tissues
  54. 54. Benefits of banking • Cord blood stem cells are not just for the baby, but may also helps the whole family • Siblings have a 25% chance of being a suitable match. • Stem cell transplants are twice as successful when the stem cells come from a family member rather than from a non-relative. (63% vs. 29%) • Once in a life time opportunity to preserve a biological resource that could be a lifesaver for the child/ other family members. • A lifesaving alternative to bone marrow transplants. • The lower probability of graft Vs. host disease (GVHD) and a greater likelihood of finding an appropriate tissue type match.
  55. 55. Cord Blood Processing 1. HLA and Infectious Disease testing. 2. Removal of RBCs 3. Addition of Dimethyl sulphoxide and Dextran. 4. Enrichment and reduction of volume to 25ml. 5. Controlled rate freezing. 6. Cryogenic storage.(Liquid nitrogen at -1960 C)
  56. 56. Process of collection and storage • Collection is same, in vaginal or in caesarean. • Can be done before or imm., after the delivery of the placenta. • Procedure is short (<10 mts) • Sterilize the umbilical cord with iodine & alcohol swabs • Sterile needle of the bag is inserted into the maternal side of the cord & blood is allowed to flow by gravity into bag. • Tubing emerging from the bag is striped, clamped and sealed/ knotted twice. • The cord blood bag is packed between the two flaps of the gel packs to maintain the temperature
  57. 57. BONE MARROW/PERIPHERAL BLOOD Umbilical CORD BLOOD Requires surgery under general anesthesia. It‟s a painful and tedious process. Obtained from the delivered placenta and umbilical cord. It‟s a painless procedure Requires a quart or more of bone marrow for transplant A few ounces can be used for transplantation. Large dose of stem cells. Rapid engraftment. Smaller dose of stem cells. Slower engraftment. After a formal search is begun, takes an average of 4 months to transplant, even if a donor is available. When a match is found, can take only few hours for the confirmatory and special tests. The fate of the recipient depends on the will of donor. Stem cells once stored is available Shelf life ranges from hours(bone marrow) to few months(peripheral blood). Long periods Latent viral infection in the donor common (CMV>50% in U.S) Rare (iCMV<1% in U.S.) Severe graft Vs. host disease (GvHD) common. Rare Generally requires a perfect match between donor and recipient for 6/6 HLA-A, -B and –DRBI antigens. Additional HLA factors (HLA-C, -DQ and –DP) increasingly used to improve prognosis. HLA mismatched cord blood transplants are possible, making it easier to find a suitable match.
  58. 58. Tissue engineering
  59. 59. Tissue engineering • Tissue engineering has broad goals including 1. Organ development 2. Elimination of waiting time for transplants 3. Creation of living tissue replacements
  60. 60. • In the past several decades, the limitations of non living mechanical solutions to organ and tissue dysfunction are now recognized and include … 1. Dialysis, 2. Mechanical heart valves, 3. Metallic orthopedic implants, 4. Non re-absorbable hernia mesh. • Current research is focused on 1. Resorbable synthetic polymers (polyglycolic acid, polyurethanes, polyglycerol) 2. Naturally occurring polymers(collagen, fibrin) 3. Minerals ( calcium triphosphate)
  61. 61. • The principal mechanical supporting structure of any engineered tissue is the SCAFFOLD. • The ideal scaffold materials for engineered tissues are Resorbable materials that break down over time. During resorption, the engineered tissue is remodeled by normal healing processes, leaving only living cellular tissue with natural supporting connective tissue. • Engineered skin substitutes were the first true clinical success of tissue engineering.
  62. 62. Key Ethical Issues • The blastocyst used in stem cell research is microscopically small and has no nervous system. Does it count as a “person” who has a right to life? • What do various religions say about when personhood begins? Does science have a view on this? • Humans Playing God ?
  63. 63. References • • pg • • • • • • • • Harrison Internal Medicine Edition 18 • Robbins Pathology Edition 8
  64. 64. !!! Thank You !!!