Therapeutic Cloning and Stem Cell Therapy

1. Therapeutic Cloning and Stem Cell Therapy Lea McLaughlin Shelby Seebacher Zach Homitz Dan Lang

3. Jamie Thomson • Born: Dec. 20, 1958 • First person to isolate and culture ESCs. • Developmental biologist and vetetarian at the university of Wisconsin at Madison. • In 2008, Time Magazine, named him one of 100 of the most influential people in the world. • 2007: Thomson's laboratory devised a method for modifying human skin cells in such a way that they appear to be embryonic stem cells without using a human embryo. They are called Induced Pluripotent Stem Cells.(IPS cells)

4. What is SCNT : Somatic Cell Nuclear Transfer? • This is also known as therapeutic cloning. • Therapeutic cloning is a technique that would be used to produce cloned embryos, but only to create embryonic stem cells that can be used to repair damaged or defective tissue in the donor of the cloned cells. • The process: • remove the nucleus of an unfertilized egg cell • replace it with the genetic material from the nucleus of a "somatic cell" (usually skin or muscle cell) • stimulate this cell to begin dividing by chemicals or electric shock • Once the cell begins dividing, stem cells can be extracted 5-6 days later and used for research

5. SCNT : Somatic Cell Nuclear Transfer • The embryonic stem cells contain the donor of the somatic cell’s DNA ;therefore, the ESC can be used to generate cells and tissues and they will not be rejected by the patient. (Who donated their somatic cell) • It is still not clear how to produce whole complex organs using this method.

6. Let’s watch

8. What are Embryonic Stem Cells?

9. What is a stem cell? • Cells with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

10. What is an embryonic stem cell? • Undifferentiated cell derived from a five day preimplantation (before the embryo implants into the uterus wall) embryo that are capable of dividing without differentiating for a prolonged period in culture and are known to develop into cells and tissues of the three primary germ layers. • Undifferentiated- a cell that has not yet developed into a specialized cell type.

11. Where are they found? Inner Cell Mass • Embryonic Stem Cells are found in the inner cell mass of a hollow blastocyst. • A blastocyst is an embryo that is 3 to 5 days old. The embryos that are used for embryonic stem cells are fertilized in vitro. 4 or 5 day old embryo.

12. How do embryonic stem cells grow outside the body? • For embryonic stem cells to grow outside the body, they must be cultured in the presence of embryonic fibroblasts. • Fibroblasts are the least specialized cells in the connective-tissues family. • Fibroblasts are needed because they provide nutrients to keep the embryonic stem cells in an undifferentiated state, and allow the cells to continue to divide. • When the cells are taken out of the presence of fibroblasts, the cell spontaneously differentiate into a cell type. • Differentiate- the process where a unspecialized cell acquires the features of a specialized cell. It is controlled by the interaction of a cell’s genes and physical and chemical condition outside the cell, usually through signaling pathways involving proteins imbedded in the cells surface. Fibroblasts

13. More on ESCs • Embryonic stem cells are cells that have almost unlimited developmental potential. They are considered pluripotent. • Pluripotent-having the ability to give rise to all cell types of the body, but not the extra-embryonic cell types. • Totipotent- having the ability to give rise to all cell types of the body, plus all the cell types of extra- embryonic cells, like the amnion, chorion and other components of the placenta.

14. Why do we need them? • Study cell differentiation • Understanding prevention and treatments of birth defects • Identify drug targets and test potential therapeutics • ESCs can make testing drugs safer. • Before, drugs were tested on animals before they were used on humans, but some drugs that tested on animal improved their condition. When tested on humans, the drugs were toxic to human heart. • Now, human ESCs that differentiate into heart cells can be tested before the drugs go to clinical trials.

15. Making an embryonic stem cell line is not very efficient. When the scientists are successful, they will carefully remove the ESCs and plate them on several different plates. They will repeat this process so that they end up with millions of ESCs. Florescent microscope picture of ESCs.

16. Advantages and Disadvantages of ESCs • Difficult to differentiate uniformly and homogeneously into a target tissue. • Capable of forming tumors or promoting tumor formation called tumorigenic. • Why? The intermolecular mechanics of stem cells are similar to the mechanics of cancerous cells. • Destruction of developing human life • Embryonic stem cells are pluriopotent, so they can form any tissue except fetus or placenta. • They can be cultured in vitro to form million of ESCs. • Will not be rejected if cell somatic cell donor is the patient.

17. More on ESCs • Embryonic stem cells have the potential to become all tissues of the body to create tissue to repair or create organs. • Embryonic stem cells are not used in clinical trials, and are still in a highly experimental stage.

18. What is an Adult Stem Cells?

19. What is it? • Adult Stem Cells are undifferentiated cells found in many organs and differentiated tissues with a limited capacity to self renew and differentiate. These cells vary in in their differentiation capacity, but is usually limited to cell types in the organ of origin.

20. What do they do in the body? • Their main role in a living organism is to maintain and repair the tissue in which they where found. • Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, fat, teeth, heart, gut, liver, ovarian epithelium, and testis. They are thought to be located in in a particular area of each tissue called a "stem cell niche". • Stem cells may remain non-dividing for periods of time until they are activated by a need for more cells to maintain tissues, disease or tissue injury. • As a baby adult stem cells are at their highest amount and as we get older, ASCs drop in number.

21. Adult Stem Cell Analogy • Everybody is born with a certain amount of stem cells, specifically, "adult stem cells." We can relate this to a "bank account”. Each person can make "withdrawals" throughout his or her life as needed. • Not all bank accounts are created equal, some people are born “with a silver spoon in their mouth” and others are born poor, but most are middle class. • Some people are able to "spend" their stem cells more than others because they have more to spend. • Environmental factors also play a key role in determining how rapidly some one’s "bank account" of (adult) stem cells is used up. However, stem cells continually diminish with age.

22. Terms • Adult stem cells can be multipotentstem cells are capable of differentiating into multiple types of cells, but not all possible types. • Multipotent-having the ability to develop into more than one cell type in the body. • Many types of adult stem cells exhibit "monopotency", meaning that they are capable of differentiating into only one particular type of cell. This "specialized" feature has typically been considered one of the major drawbacks of adult stem cells. • Monopotency- Cells capable of differentiating into only one particular type of cell. This "specialized" feature has been considered one of the drawbacks of adult stem cells.

23. Transdifferentiation • Experiments have been reported that certain adult stem cell types can differentiate into cell types seen in organs or tissues other than those expected from the cells' predicted lineage • Example- • brain stem cells that differentiate into blood cells. • blood-forming cells that differentiate into cardiac muscle cells.

24. Differentiation of different stem cells • Hematopoietic stem cells give rise to all the types of blood cells • Mesenchymal stem cells give rise to a variety of cell types: bone cells, cartilage cells, fat cells, and other kinds of connective tissue cells such as those in tendons. • Neural stem cells in the brain give rise to its three major cell types: nerve cells (neurons) and two categories of non-neuronal . Hematopoietic stem cell

25. Differentiation of different stem cells • Epithelial stem cells in the lining of the digestive tract occur in deep crevasses and give rise to several cell types. • Skin stem cells occur in the basal layer of the epidermis and at the base of hair follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to the epidermis.

26. How to collect Adult Stem Cells? • It depends on the location of where the adult stem cells are extracted from. • Ex. Peripheral blood stem cells (PBSCs) are collected by an apheresis. • Apheresis- a process in which the donor is connected to a special cell separation machine by a needle inserted in the vein. • Blood is taken from one vein and is circulated through the machine which removes the stem cells and returns the rest of the blood and plasma back to the donor through another needle inserted into the opposite arm.

27. How to collect Adult Stem Cells • Ex.2 • Collecting bone marrow involves gathering stem cells with a needle placed into the soft center of the bone marrow. Most sites where bone marrow is collected is located in the hip bones and the sternum.

28. Advantages • Adult stem cells are ethically and politically noncontroversial. • Special adult-type stem cells from bone marrow and from umbilical cord have been isolated recently which appear to have high plasticity as the embryonic type • Plasticity-the ability of an adult stem cell from one tissue to generate the specialized cell type or types of another tissue • Already somewhat specialized so inducement may be simpler. • Not immunogenic—recipients who receive the products of their own stem cells willnot experience immune rejection. • Some adult stem cells are easy to gather(skin,muscle, marrow, fat), while others may be more difficult to obtain (brain stem cells). The umbilical and placental stem cells are readily available. • Non-tumorigenic—do not usually form tumors • No harm done to the donor

29. Disadvantages • Can sometimes be difficult to find in large numbers. • May not live as long as ES cells in culture • Low Plasticity - (with the exception bone marrow and umbilical cord)—may be more difficult to reprogram toform other tissue types. • Once cultured, the cells are slow to grow.

30. ESCs vs. ASCs -Both differentiate into more specialized cell type

31. Stem Cell Therapy Used Today

32. Bone Marrow • Bone Marrow transplant is the most well known. • Bone marrow contains hematopoietic stem cells which produce blood and immune cells. • Used to treat leukemia or blood or immune system diseases • Existing bone marrow may have to be killed by chemotherapy or radiation.

33. Peripheral Blood stem cells • Small number of hematopoietic stem cells circulate in the bloodstream. • It is a less invasive procedure. • Collecting enough hematopoietic stem cells is difficult because of the small number.

34. Umbilical Cord Blood • Cord blood contains hematopoietic stem cells. These hematopoietic stem cells are usually called neonatal stem cells. • They are less mature than those stem cells found in the bone marrow. • Rejected less often because they have not developed proteins that can be recognized by patients immune system. • Lack well developed immune cells, so they are less likely to attack recipient’s body-graft versus host disease.

35. Types Transplants • In autologous transplants, patients receive their own stem cells. • In syngeneic transplants, patients receive stem cells from their identical twin. • In allogenic transplants, patients receive stem cells from their brother, or parent or person who is not related to the patient .

36. Skin • Used on patients who suffered from extensive burn damage. • Skin was made using stem cells from bone marrow. • Once on the patient the dermal layer begins to regenerate and stem cells were differentiated into skin cells. • Now skin stem cells found on a hair follicle. • stem cells in the hair follicle receive a signal to divide therefore a round of hair growth begins.

38. Adult stem cells used to create windpipe • Scientist implanted a new windpipe into a woman whose airway was badly damaged by tuberculosis. • Doctors took adult stem cells from the bone marrow of Claudia Castillo. • Once in laboratory dishes scientists injected various chemicals to induce the cells to turn into highly specialized cells. • Scaffold-a trachea , a portion of the windpipe, taken from a 51-year-old man who died. The trachea was rinsed with antibiotics, and the cells were removed with enzymes. • The stem cells coated the outside and inside of the trachea. • They applied nutrients and chemicals to promote the growth of new layers of tissues.

40. Current Experimental Procedures

41. Experiments Using Adult Stem Cells • Horses-stem cells are used to heal tendons and ligaments. • The stem cell used is mesenchymal. (come from bone marrow, umbilical cord, and fat tissue) -James Andrew Lee, M.D., was the first researcher to demonstrate in vivo(in the body) that exposing adult stem cells derived from fat to different growth factors to generate bone, cartilage and other cells.

42. Dental stem cells • Dental adult stem cells were put in the hippocampal areas of mice. Hippocampal is a part of the brain that play an important roles in long term memory and spatial navigation. These cells started the growth of new neural cells.(many of these formed neurons) • More potential for adult stem cells

43. Spinal Cord injury • In a mouse, grafting human neural stem cell(adult stem cells) in the spine, the cell differentiate appropriately and help mice to recover. Human neural cells

44. Insulin like Growth Factor • Uses an insulin like growth factor(IGF) • Promotes muscle repair • Experiment using a mouse

45. IPS(Induced Pluripotent Stem Cells)

46. What is it? • IPS cells are mature cells that have been genetically reprogrammed to an embryonic stem cell like state. • IPS cells are very similar to ESCs. • Both cells a had round shape, a large nucleus and cytoplasm. Colonies of IPS cells were also similar to that of ESCs. • IPS cells divide at a rate equal to ESCs. • IPS cells expressed cell surface antigenic markers expressed on ESCs. • Express same genes • Both have high telomerase activity.

47. Why are they useful? • IPS cells are valuable tools for drug development and modeling of diseases, and scientist hope to use them in transplantation medicine.

48. How do you make them? • Viruses are used to introduce the reprogramming factors into adult cells. • This process must be carefully controlled and tested before it can be used on humans. In animal studies, the virus used to introduce stem cell factors sometimes cause cancers. • Researchers are looking for a non-viral way to introduce stem cell factors.

49. Viral vectors • In 2006, Shinya Yamanaka used retroviruses to insert four genes into the chromosome of mouse skin cells. This successfully turned the adult stem cells to IPS cells, which could become any of the body tissues. • The following year he redid his experiment with human cells. He was able to make IPS cells, but it was too risky to take cells grown from IPS cells and transplant them into a human. • The genes(used to reprogram) themselves are known to trigger cancer. Also when retroviruses land in the chromosome, they can disrupt genes that normally keep cancers from forming.

50. Genes Used to Convert Cells • Oct4 and Sox2 play an important role in the maintenance of embryonic stem cell pluripotency. • Myc and Klf4 are the two other genes scientists have discovered that are necessary to convert somatic cells to IPS cells.