Summer 2007 Workshop in Biology and Multimedia for High School Teachers

1. Summer 2007 Workshop • in Biology and Multimedia • for High School Teachers

2. Stem Cell Research Overview Mountainous Path

3. Outline • What are Stem Cells? • Potential Uses • Claims Against Using Stem Cells • Cultivation Process • Stem Cells and Cloning • Stem Cell Theory of Cancer • Worldwide Status

4. What are stem cells? • Stem cells are undifferentiated cells that have many potential scientific uses: • Cell based therapies • Often referred to as regenerative or reparative medicine • Therapeutic cloning • Gene therapy • Cancer research • Basic research

5. Two types of stem cells • Embryonic Stem Cells (ESC): received from: • Embryos created in vitro fertilization • Aborted embryos • Adult Stem Cells (ASC): can be received from: • Limited tissues (bone marrow, muscle, brain) • Discrete populations of adult stem cells generate replacements for cells that are lost through normal wear and tear, injury or disease • Placental cord • Baby teeth

6. Source of ESC • Blastocyst • “ball of cells” • 3-5 day old embryo • Stem cells give rise to multiple specialized cell types that make up the heart, lung, skin, and other tissues • Human ESC were only studied since 1998 • It took scientists 20 years to learn how to grow human ESC following studies with mouse ESC

7. How are embryonic stem cells harvested? • Human ES cells are derived from 4-5 day old blastocyst • Blastocyst structures include: • Trophoblast: outer layer of cells that surrounds the blastocyst & forms the placenta • Blastocoel: (“blastoseel”) the hollow cavity inside the blastocyst that will form body cavity • Inner cell mass: a group of approx. 30 cells at one end of the blastocoel: • Forms 3 germ layers that form all embryonic tissues (endoderm, mesoderm, ectoderm)

8. Blastocyst http://www.ivf-infertility.com/infertility/infertility5.php

9. Unique characteristics of Stem Cells • Stem cells can regenerate • Unlimited self renewal through cell division • Stem cells can specialize • 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

10. Unspecialization • Stem Cells are unspecialized • 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)

11. Self - Renewal (Regeneration) • Stem cells are capable of dividing & renewing themselves for long periods • This is unlike muscle, blood or nerve cells – which do not normally replicate themselves • In the lab, a starting population of SCs that proliferate for many months yields millions of cells that continue to be unspecialized • These cells are capable of long-term self-renewal

12. Specialization of Stem Cells: Differentiation • 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

13. Differentiation • Why do your body cells look different although they all carry the same DNA, which was derived from one fertilized egg? • Differentiation example (http://learn.genetics.utah.edu/units/biotech/microarray/)

14. Potential of Stem Cells (vocab) • Totipotent (total): • Total potential to differentiate into any adult cell type • Total potential to form specialized tissue needed for embryonic development • Pluripotent (plural): • Potential to form most or all 210 differentiated adult cell types • Multipotent (multiple): • Limited potential • Forms only multiple adult cell types • Oligodendrocytes • Neurons

15. Adult Stem Cells • Adult or somatic stem cells have unknown origin in mature tissues • Unlike embryonic stem cells, which are defined by their origin (inner cell mass of the blastocyst)

16. http://www.stemcellresearch.org/testimony/20040929prentice.htm Reprinted with permission of Do No Harm.

17. Adult stem cells continued • Adult stem cells typically generate the cell types of the tissue in which they reside • Stem cells that reside in bone marrow give rise to RBC, WBC and platelets • Recent experiments have raised the possibility that stem cells from one tissue can give rise to other cell types • This is known as PLASTICITY

18. Adult Stem Cell Plasticity Examples • Blood cells becoming neurons • Liver cells stimulated to produce insulin • Hematopoietic (blood cell producing) stem cells that become heart cells • CONCLUSION: Exploring the use of adult stem cells for cell-based therapies has become a very important (and rapidly increasing) area of investigation by research scientists!

19. Adult stem cells: A brief history • Adult stem cell research began about 40 years ago • Stem cell discoveries in 1960s: • 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

20. History Cont. • Stem Cell Discoveries in the 1990s • Neural stem cells in brain are able to generate the brain’s three major cell types • Astrocytes • Oligodendroglial cells • Neurons http://www.alsa.org/images/cms/Research/Topics/cell_targets.jpg

21. Adult Stem Cell Facts • Adult stem cells were found in many more tissues than expected • Some may be able to differentiate into a number of different cell types, given the right conditions • General consensus among scientist: • 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

22. 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

23. Potential uses cont. • 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

24. Potential uses cont. • Cell based therapies: • 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

25. Embryonic vs Adult Stem Cells • Totipotent • 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 cell types, limited outcomes • Unknown source • Limited numbers, more difficult to isolate • Less likely to cause immune rejection, since the patient’s own cells can be used

26. Claims against ESC (unsubstantiated thus far!) • Difficult to establish and maintain * • Difficulty in obtaining pure cultures from dish* • Potential for tumor formation and tissue* destruction • Questions regarding functional differentiation • Immune rejection • Genome instability • Few & modest results in animals, no clinical treatments • Ethically contentious * = same problem with ASC

27. Cell Culture Techniques for ESC • Isolate & transfer of inner cell mass into plastic culture dish that contains culture medium • Cells divide and spread over the dish • Inner surface of culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide

28. This coating is called a FEEDER LAYER Feeder cells provide ES cells with a sticky surface for attachment Feeder cells release nutrients Recent discovery: methods for growing embryonic stem cells without mouse feeder cells Significance – eliminate infection by viruses or other mouse molecules ES cells are removed gently and plated into several different culture plates before crowding occurs

29. http://www.news.wisc.edu/packages/stemcells/illustration.html Images depict stem cell research at the University of Wisconsin Madison.

30. Cloning of whole organisms • Purpose: • Reproductive cloning in animals • Therapeutic cloning in animals • Breeding animals or plants with favorable traits • Producing TRANSGENIC animals that: • Make a therapeutic product (vaccine, human protein etc) • Act as animal models for human disease • Deliver organs that will not be rejected (cells lacking cell surface markers that cause immune rejection) • Vaccines in biotech industry: steps in cloning a gene

31. SCNT: Somatic Cell Nuclear Transfer • SCNT is a method used for: • Reproductive cloning such as cloning an embryo • Regenerative cloning to produce “customized” stem cells & overcome immune rejection • Blastula stage cannot continue to develop in vitro • It must be implanted into surrogate mom • Surrogate mom is just a container that provides protection & chemical signals necessary for development

32. http://www.kumc.edu/stemcell/early.htmlReprinted with permission from the University of Kansas Medical Center.

33. http://www.stemcellresearch.org/testimony/20040929prentice.htm Reprinted with permission of Do No Harm.

34. Challenges of Reproductive Cloning • Many animals were cloned after Dolly • Cats, pigs, mice, goats, cattle, rabbits • Obstacles: • Very inefficient process • Most clones have deleterious effects & die early • Surviving clones show premature aging signs • Signs of abnormal embryonic development: • Clones & their placentas grow much faster than expected in surrogate mom

35. Therapeutic Cloning • 3 goals of therapeutic cloning by SCNT in humans: • Use embryo as source for ES cells • Use ES cells to generate an organ • In this case the organ generated will carry cells with the same genetic markers as the patient (recipient) • Correct genetic error in ESC in blastula stage

36. Pitfalls of therapeutic cloning (1)Some immune rejection may occur- WHY? • About 1% of the DNA in the clone will NOT be identical to donor cell (patient) • It will be identical to egg cell used in SCNT • REASON: mitochonrial DNA in eggs • Human mitochondria carry about 13 genes, some of which code for surface proteins

37. Pitfalls of therapeutic cloning (2) • Large number of eggs needed for SCNT • To harvest large number of eggs: • Excessive hormone treatment of females to induce high rate of ovulation • Surgery to retrieve eggs • Both can be harmful to female human • Cow/pig females may be used • Cow/pig eggs will carry species-specific mitochondrial genes • Mixing species is reason for concern!

38. Common Opinions • Reproductive cloning is a criminal offense (it is ILLEGAL worldwide!) • Therapeutic cloning is acceptable, however there is still significant controversy over whether: the clone is implanted into the uterus of surrogate mom? OR the clone is explanted into culture dish to generate ES cells

39. Stem Cell Theory of Cancer • 1855: Rudolf Virchow developed the Embryonal- Rest Hypothesis • Microscopic examination of tumor samples revealed many morphological (structural & functional) resemblances to ESC in a developing fetus • Isolation of teratoma: nonmalignant tumors • Teratoma represents a ball of almost all cell types • This indicates that teratoma may originate from unregulated stem cells that can give rise to almost all tissues

40. Teratoma • Ovarian Teratoma • You can see teeth! http://home.earthlink.net/~radiologist/tf/040802.htmImage courtesy of Leonard J. Tyminski, M.D., Radiologist at earthlink.net

41. Tumor stem cell Tumor cell Current Efforts with SC and Cancer • Determine difference between cancer & normal stem cells • Identify potential points in pathways critical for the survival of cancer SCs • Develop therapies that specifically target cancer SC • Duke University Explanation Drawn by Christine Rodriguez

42. Status of SC research in other countries • Great Britain • Very liberal policies on research • Therapeutic cloning allowed, use of excess embryos & creation of embryos allowed • Stem cell research allowed • France • Less liberal politics • Use of excess embryos from IVF allowed • Reproductive AND therapeutic cloning banned • Germany • Very strict policies • Use of excess embryos and creation of embryos banned • Scientists can IMPORT embryos

43. Debate in US • Federal funding available for research using the Bush lines only: • ES cell lines that were already in existence by 8/9/01 • Disadvantage of Bush stem cell lines: • May have lost regenerative ability • May have accumulated mutations or infections • Private companies continue to pursue stem cell research • Use of human embryos for IVF & therapeutic cloning is legal in most states • No federal funding • Some states are considering banning both

44. Global Status • Ongoing debate regarding use of embryos • United Nations: proposal for a global policy to ban reproductive cloning only

45. References • Stem cells & Cloning Stem cells & Cloning; David A. Prentice, Benjamin Cummings, 2003 • http://www.pbs.org/wgbh/nova/sciencenow/3302/06.html • http://www.stemcellresearch.org • http://www.stemcells.nig.gov/info/nasics/nasics7.asp • http://www.stemcells.nig.gov/info/scireport/2006report.htm • http://www.whitehouse.gov/news/re;eases/2001/08/20010809-2.html • Stem cells in class; Badran, Shahira; Bunker Hill Community College, 2007, Boston Museum of Science Biotechnology Symposium • Harvard Stem Cell Institute