1. By Dept. of Orthopaedic Surgery C.S.M.M.U., Lucknow

2. Introducing stem cells A life story starts with single stem cell…the Zygote- embryonic stem cells inside blastocyst - a very early embryo Trophectoderm

3. Location of Adult Stem Cells Adult stem cells and progenitor cells reside through out your body These stem cells reside in a specific area of each tissue called the “stem cell niche” This niche is a particular microenvironment that fosters the growth of resident stem cells Mutations in cells, signals they receive, and changes in the microenvironment can activate a stem cell

4. Adult stem cells: Where we find them muscles skin surface of the eye brain breast intestines (gut) bone marrow testicles

5. Stem cells are unspecialized cells Stem cells can differentiate and become another type of cell with a specialized function Stem cells replenish without limit Stem cells act as a repair system to the body Stem cells are being researched because they have the unique ability to replace damaged cells with healthy cells Scientists believe research will result in treatments and cures for diseases.

6. Unique characteristics of Stem Cells Self renewal a stem cell can divide (renew itself) indefinitely (go through mitosis) and without always developing into another cell Plasticity A stem cell is plastic, meaning it can develop into another type of cell Stem cells can specialize/differentiate Under certain physiologic or experimental conditions – Stem cells then become cells with special functions such as: Beating cells of the heart muscle Insulin-producing cells of the pancreas

7. Unspecialization of stem cell – They do not have any tissue-specific structures that allow for specialized function – Stem cells cannot work with its neighbors to pump blood through the body (like heart muscle cells) – They cannot carry molecules of oxygen through the bloodstream (like RBCs) – They cannot fire electrochemical signals to other cells that allow the body to move or speak (like nerve cells)

8. Specialization of Stem Cells Differentiation: unspecialized stem cells give rise to specialized (differentiated) cells in response to external and internal chemical signals – Internal signals: turn on specific genes causing differential gene expression – External signals: include: Chemicals secreted by other cells such as growth factors, cytokines, etc Physical contact with neighboring cells Extrinsic factor(s)

9. Particular specialized cells DIFFERENTIATION (specializing) stem cell pool stem cell Committed Progenitors SELF-RENEWAL (copying) Tow ways of stem cell division 1. Simple cell divisions

10. DIFFERENTIATION (specializing) specialized cell e.g. muscle cell, nerve cell committed progenitors: Plasticity 2. stem cell

11. Two Conceptual Views of Stem Cells

12. Potential types of Stem Cells Totipotent -cells produced by the first few divisions of the cell ( cells of 1-3 days embryo). So can form any cell of the embryo as well as the placenta.. Pluripotent – these cells differentiate into cells derived from the three germ cell layers ( cells from 5 to 14 days old embryo). Eg: embryonic stem cell, embryonic germ cell and embryonic carcinoma cells. Multipotent – Limited potential – Forms only multiple adult cell types e.g. cord blood, bone marrow stem cells, testicular stem cells etcOligopotent – these cells can produce cells of a closely related family of cells. Eg: haematopoeitic stem cells, neural and mesenchymal stem cells Unipotent – these cells only produce one cell type., but have the property of self renewal which distinguishes them from the non stem cells.

14. Types of stem cell (origin based): 1)Embryonic stem cells Totipotent: undifferentiated cells derived from zygote to 1-3 days post fertilization Cells of inner cell mass of blastocyst, Pluripotent:Cells of inner cell mass of blastocyst, Cells of any germ layer 2) Adult/Tissue stem cells- undifferentiated cell(s) found in a differentiated tissue(s) Located in few organs Multipotent: Cells from various specific sites Located in few organs 3) Induced pluripotent stem cells (iPS) ‘ ‘genetic reprogramming’ = add certain genes to the cell Adult stem cell iPS-behaves like a embryonic pluripotent cell

15. Embryonic vs Adult Stem Cells Embryonic vs Adult Stem Cells Totipotent or pluripotent – Differentiation into ANY cell type Known Source Large numbers can be harvested from embryos May cause immune rejection – Rejection of ES cells by recipient has not been shown yet Multi or pluripotent -Differentiation into some specific cell types i.e. limited outcomes Located in few organs or may be unidentified Limited numbers, more difficult to isolate Less likely to cause immune rejection, since the patient’s own cells can be used

16. Derived from the inner cell mass/epiblast of the blastocyst. Capable of undergoing an unlimited number of symmetrical divisions without differentiating (long-term self-renewal). Exhibit and maintain a stable, full (diploid), normal complement of chromosomes (karyotype). Capable of integrating into all fetal tissues during development. Lacks the G1 checkpoint in the cell cycle. Spend most of their time in the S phase of the cell cycle, during which they synthesize DNA. It do not require any external stimulus to initiate DNA replication. Do not show X inactivation. Fundamental properties of an Embryonic Stem Cell

17. Stages of Early Embryonic Development

18. Two Sources of Embryonic Stem Cells 1.Excess fertilized eggs from IVF (in-vitro fertilization) clinics 2.Therapeutic cloning (somatic cell nuclear transfer)

19. SCNT can lead to either reproductive or therapeutic cloning

20. embryonic stem cells taken from cell mass culture in the lab to grow more cells fluid with nutrients cells inside = ‘inner cell mass’ ES cell culture

21. Simple differentiation all possible types of specialized cells skin grow under conditions A neurons grow under conditions B blood grow under conditions C liver grow under conditions D

22. ES cell differentiation example

23. Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment. These surplus embryos can be used to produce stem cells. Regenerative medical research aims to develop these cells into new, healthy tissue to heal severe illnesses.

24. Somatic Cell Nuclear Transfer The nucleus of a donated egg is removed and replaced with the nucleus of a mature, "somatic cell" (a skin cell, for example). No sperm is involved in this process, and no embryo is created to be implanted in a woman’s womb. The resulting stem cells can potentially develop into specialized cells that are useful for treating severe illnesses. used to overcome rejection barriers

25. Cloned Embryonic Stem Cells – Advantages/Problems Advantages – No rejection – “Prefect match” Problems – Only 10% of cloned oocytes became embryos – 0% (0 out of 2061) survived to become a cell line – Genetic donor was same as egg donor (i.e., won’t work for males!) – Cost is high

26. Unknowns in Stem Cell/Cloning Research It is uncertain that human embryonic stem cells in vitro can give rise to all the different cell types of the adult body It is unknown if stem cells cultured in vitro (apart from the embryo) will function as the cells do when they are part of the developing embryo Stem cells need to be differentiated to the appropriate cell type(s) before they can be used clinically

27. Recently, abnormalities in chromosome number and structure were found in three human ESC lines Stem cell development or proliferation must be controlled once placed into patients Possibility of rejection of stem cell transplants as foreign tissues is very high

28. Adult stem cell sources/regions of body Identified in many organs and tissues - Adult stem cells have been isolated from amniotic fluid, umbilical cord blood, umbilical cord, peripheral blood, Blood vessels, Bone marrow, brain tissue, Skeletal muscle, liver, pancreas, cornea, salivary gland, skin, adipose tissue, heart, cartilage, thymus, dental pulp, Gut, Ovarian epithelium and Testis.

29. Adult Stem Cell Facts Adult or somatic stem cells have unknown origin in mature tissues Adult stem cells typically generate the cell types of the tissue in which they reside – e.g. Bone marrow contains 2 populations of stem cells Hematopoietic stem cells – forms all blood cell types Bone marrow stromal cells – mixed cell population that generates bone, cartilage, fat and fibrous connective tissue – Rat brain contains two regions of dividing cells, which become nerve cells Adult stem cells were found in many more tissues than expected

30. Adult stem cell differentiation MULTIPOTENT blood stem cell found in bone marrow and differentiation only specialized types of blood cell: red blood cells, white blood cells, platelets Number of cell divisions

31. Types of Adult Stem Cells  Hematopoietic stem cells: blood and immune system  Mesenchymal stem cells: bone, cartilage, fat, muscle, tendon/ligament  Neural stem cells: neurons, glial cells  Epithelial & Endothelial stem cells: skin, linings Some other’s are-Mammary, Olfactory, Testicular etc

32. Dr. Said M. Aj. Shawar, Biotechnology - AGU 32 Umbilical cord (UC) stem cells -Obtained from UC immediately after birth -Like BM, UC blood is a good source for hematopoietic (HP) stem cells. -Called neonatal stem cells -Less mature than HP cells in adults. -Easily obtained and abundant.

33. Hematopoietic stem cells Give rise to all the blood cell types: Myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) Lymphoid (T-cells, B-cells, NK-cells) Found in the bone marrow from very early on in development, as well as in umbilical cord blood and placental tissue

34. Neural stem cells They are located in: Subventricular zone lining the lateral ventricles, where they give rise to newly-born neurons that migrate to the olfactory bulb via the rostral migratory stream Subgranular zone, part of the dentate gyrus of the hippocampus Neural stem cells (also called Neural precursor cells) give rise to neurons, oligodendrocytes, and astrocytes Top: Section of the hippocampus, blue dots are neural stem cells Left: Mature neuron (red) © CIRM

35. Mesenchymal stem cells These stem cells will differentiate into: – cartilage cells (chondrocytes) – muscle cells (myocytes) – fat cells (adipocytes) – tendons, ligaments, and connective tissue (epithelial cells including osteoblasts) These cells are located throughout the body Bone marrow, fat, and cord blood are easiest to isolate Smooth muscle cells (red) © CIRM

36. Epithelial stem cells Give rise to epithelial cells which constitute 60 percent of the differentiated cells in the body. Responsible for covering the internal (i.e. intestinal lining) and external surfaces (i.e. skin) of the body, including the lining of vessels, glands, and other cavities. Epithelial stem cells are also found in the bulge region of the hair follicle Retinal pigment epithelial cells

37. Cells in suspension are tagged with fluorescent markers specific for undifferentiated stem cell Labeled cells are sent under pressure through a small nozzle and pass through an electric field A cell generates a negative charge if it fluoresces and a positive charge if it does not. Laser beam passes through one cell FLUROSCENT ACTIVATED CELL SORTING Stem cell SEPARATION OF STEM CELL

38. Adult stem cells DO NOT have as much potential as embryonic stem cells CLARIFICATION: not all new adult cells arise from stem cells-Most arise by MITOSIS of differentiated cells Recent experiments have raised the possibility that stem cells from one tissue can give rise to other cell types This is known as PLASTICITY e.g, - Blood cells becoming neurons -Hematopoietic (blood cell producing) stem cells that become heart cells -Liver cells stimulated to produce insulin General consensus of ASCs among scientist:

39. Features of Adult Stem Cells ● Adult stem cells migrate throughout the body in the blood. ● Reach and bind to specific site ● Switch into specific tissues ● You can harvest adult stem cells from the individual to be treated. Therefore, there are no rejection problems

40. Benefits of Adult Stem Cell Research Easy to obtain Potentially limitless in supply Patients can use their own stem cells for treatment and therapy Adult stem cells are politically neutral Not offensive to any major interest group nor do they generate controversy.

41. Limitations of adult stem cell The isolation of some types of ASC, for example the isolation of neural cells from a patient's brain, would be impractical Where a person suffers from a genetic disorder or some types of cancers, ASC isolated from that individual will retain the damaging genetic alterations underlying the disease and so be of little therapeutic value Unambiguous identification is difficult Maintenance in culture is difficult

42. Potential of Adult Stem Cells

44. Induced pluripotent cell Culture medium Stem cells can be grown in liquid culture medium that contains: –Nutrients to feed the cells. –Hormones –Growth factors –Signaling molecules –Etc. Induced Plasticity of Adult Stem Cell- - Blood cells becoming neurons - Liver cells stimulated to produce insulin - Hematopoietic (blood cell producing) stem cells that become heart cells

45. History of Human Stem Cell Research 1954 – John Enders received a Nobel prize in Medicine for growing polio virus in human embryonic kidney cells In 1968, the first bone marrow transplant was successfully used in treatment of SCID Since the 1970’s, bone marrow transplants have been used for treatment of immunodeficiencies and leukemias

46. In 1998, James Thomson (University of Wisconsin-Madison) isolated cells from the inner cell mass of the early embryo, and developed the first human embryonic stem cell lines. In 1998, John Gearhart (Johns Hopkins University) derived human embryonic germ cells from cells in fetal gonadal tissue (primordial germ cells). Pluripotent stem cell “lines” were developed from both sources

47. Early Successes – Adult Stem Cells Human mesenchymal stem cells can produce cells of bone, cartilage, adipose, muscle, hematopoiesis-supporting stromal, endothelial, and neuronal cells. Some multipotent adult progenitor cells have been shown to differentiate into functional, hepatocyte-like cells. Human neural stem cells can migrate extensively in the brain after injection 2001 – First cloned human embryos (only to six cell stage) created by Advanced Cell Technology (USA) 2004* – Claim of first human cloned blastocyst created and a cell line established (Korea) – later proved to be fraudulent

48. Regenerative Medicine Process of creating living, functional tissues to repair or replace tissue or organ function lost due to age, disease, damage, or congenital defects It helps to produce extended healthy longevity,

49. Possible Uses of Stem Cell Technology Possible Uses of Stem Cell Technology Replaceable tissues/organs Repair of defective cell types Therapeutic issues  Delivery of genetic therapies  Delivery chemotherapeutic agents It provides an ideal model for the study of development of organisms It also aids in drug discovery

50. Potential Uses of Stem Cells Basic research – clarification of complex events that occur during human development & understanding molecular basis of cancer – Molecular mechanisms for gene control – Role of signals in gene expression & differentiation of the stem cell – Stem cell theory of cancer Biotechnology (drug discovery & development) – stem cells can provide specific cell types to test new drugs – Safety testing of new drugs on differentiated cell lines – Screening of potential drugs Cancer cell lines are already being used to screen potential anti- tumor drugs Availability of pluripotent stem cells would allow drug testing in a wider range of cell types & to reduce animal testing

51. Regenerative therapy to treat Parkinson’s, Alzheimer’s, ALS, spinal cord injury, stroke, severe burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis Stem cells in gene therapy Stem cells as vehicles after they have been genetically manipulated Stem cells in therapeutic cloning Stem cells in cancer Cell based therapies:

52. Future Applications Stem Cells may one day help scientists to regenerate cells lost in diseases like: – Repair heart muscle after a heart attack – Pancreas cells lost in diabetes – Neurons lost in Alzheimer’s – Retinal cells causing blindness – Understand the cell growths of cancers – Help organ transplantation Clinical trials are already underway using bone marrow (adult) stem cells for treatment of heart attacks, liver disease, diabetes, bone and cartilage disease and brain disorders.

53. Treating Leukemia Leukemia results when white blood cells made in bone marrow begin to grow and function abnormally One way to do this is through chemotherapy When chemotherapy cannot solve the problem, doctors turn to bone marrow transplants.

54. Another Stem Cell Victory - Parkinson's Disease A degenerative disorder of the central nervous system Sufferers lack a sufficient amount of a brain chemical called dopamine The cure of this disease is to multiply cells that release dopamine

55. Insulin Independence There is insulin independence for Type 1 diabetes patients A study showed patients receiving injections with adult stem cells were able to go as long as four years without having to rely on insulin shots.

56. Healing of Heart by Adult Stem Cells Cardiologists and heart surgeons worldwide currently use adult stem cell therapy to treat patients suffering from coronary artery disease, cardiomyopathy, and congestive heart failure.

57. Process of stem cells for spinal cord injury Bone Marrow taken from the hip 6 weeks of cultivating the adult stem cells to increase total to 100 million. Adult Stem Cells injected at injury site (C3-C4) in a surgical procedure

58. Thalesemia The genetic defect results in reduced rate of synthesis of one of the globin chains that make up hemoglobin Hematopoietic stem cell transplantation (HSCT) is the only curative approach

60. Types of stem cell for transplantation Autologous adult Allogeneic adult Foetal (cord blood) Embryonic Mesenchymal

61. Mesenchymal stem cells injected into rat heart increased pumping capacity and vessel growth after heart attack. – Journal of Clinical Investigation 115:326–338, 2005. “Stembrids” were made —one ESC was enucleated and then given the nucleus from an adult somatic cell. – Not shown that the resulting “stembrid” would be immunologically acceptable to the adult somatic cell donor. Recent Research

62. Scientists have a lot of work to ID different SC