Announcements

1. Announcements • Monday, April 23: genomic equivalence, cloning, and stem cells: pp. 727-730 • Wednesday, April 25: cell biology of cancer, pp. 762-770, 775-789 • Friday, April 27, 9 AM: optional review session • Monday, April 30, 8 AM: final exam • 139 points; 90 points from days 34-43; 49 points cumulative (days 1-43).

2. Cloning Embryonic Stem cells After reading the text, attending lecture, and reviewing lecture notes, you should be able to: Describe the potential fates of stem cells in general. Distinguish reproductive cloning from therapeutic cloning. Describe how embryonic stem cells are obtained, how they are assayed for potency, and how they can be used. Outline/Learning objectives

3. Cloning Mammals: Evidence of nuclear equivalence and reprogramming Black (ooplasm donor) Agouti (nucleus donor) Albino (nucleus donor) Cloned mice (Agouti) ^ Cloned mice (b. 1998) and their “parents.” Coat color is used as a marker. Conclusion: Some differentiated nuclei can be completely reprogrammed. Dolly (July 5, 1996 - Feb. 14, 2003) and one of her 6 lambs Wilmut et al. 1997. Nature 385:810-814.

4. Two ways to make an embryo:1. Sexual reproduction Source: Human Cloning and Genetic Modification: The Basic Science You Need to Know, Association of Reproductive Health Professionals, www. arhp.org

5. Two ways to make an embryo:2. Cloning or asexual reproduction “Nuclear Transplantation” Source: www. arhp.org

6. Reproductive cloning Goal is to produce a clonal embryo to implant in a mother’s womb with intent to carry the child to birth. Therapeutic cloning Goal is to produce a clonal embryo to generate embryonic stem cells. ES cells can be used to treat clonal donor without risk of immune rejection. Human Reproductivevs. Therapeutic Cloning

7. Top 10 causes of U.S. deaths (1998) Cause# (1000’s)#/100,000% • Heart disease 724 268.2 31.0 • Cancer 541 200.3 23.3 • Stroke 158 58.6 6.8 • Chronic lung disease 158 58.6 6.8 • Accidents 98 36.2 4.2 • Pneumonia/influenza 92 34.0 3.9 • Diabetes 65 24.0 2.8 • Suicide 31 11.3 1.3 • Kidney disease 26 9.7 1.1 • Chronic liver disease 25 9.3 1.1 Tissue damage, e.g. spinal cord injury, also potentially treatable with stem cells.

8. Study Suggests New Avenue on Diabetes By THE ASSOCIATED PRESS Published: April 11, 2007CHICAGO, April 10 (AP) — Thirteen young diabetics in Brazil have been able to stop receiving insulin after being treated with stem cells taken from their own blood, researchers are reporting.

9. What’s a stem cell? • Stem cells are defined by their ability to: • Continue to grow and divide • Given the right signals (e.g. hormones or growth/differentiation factors), to differentiate into a specialized cell type • Stem cells have different potencies: • Unipotent: makes one cell type • Multipotent: makes several cell types • Pluripotent: makes most cell types • Totipotent: makes all possible cell types (zygote)

10. Where do stem cells come from?

11. Evidence for ES cell pluripotency

12. Evidence of adult stem cell pluripotency

13. But: the pleuropotency of adult stem cells may be exaggerated • Many such studies have not been replicated. • In contrast, the totipotency of embryonic stem cells is a fact. • However, the previously-approved lines have dwindled and newer, better lines are not allowed. • The Senate approved another stem cell bill 63-34. President Bush has vowed to veto it again.

14. Stem Cell Therapeutics

15. Some Stances on Human Cloning and Stem Cell Research • Support any human cloning • Few biomedical scientists • Clonaid (Raelians - Bahamas), Severino Antinori (Italy), Panos Zavos (Kentucky) • Oppose reproductive cloning, support therapeutic cloning • Many biomedical scientists • Hatch-Feinstein-Specter-Kennedy et al. bill S.303 in U.S. Senate • Nancy Reagan • Oppose any human cloning • Right-to-life groups: destruction of embryo, slippery slope • Brownback-Landrieu et al. bill S.245 in U.S. Senate • President Bush

16. ES cells in the news: The mostspectacular fraud in biomedical research • What three accomplishments did Dr. Hwang Woo Suk claim? • First line of human embryonic stem cells generated from nucleus of adult cell > first step in therapeutic cloning (2004). FRAUD • Improvement in efficiency of above, generating ES cell lines from 9 patients > practical treatment through therapeutic cloning (Science 2005. 308:1777-1783). FRAUD • First cloning of a dog (Nature 2005. 436:641). VALIDATED • What ethical breaches apparently occurred in his laboratory? • Junior researchers were compelled to donate eggs for research in his lab. • Payments were made to egg donors through a Seoul hospital. • He lied about it this knowledge to the journal Science. • Dr. Woo Suk ordered subordinates to falsify data.

17. Korean Stem Cell Scandal, 2005-2006 (Hwang et al. 2005) Duplicated Duplicated Cropped Cropped Cropped

18. Cancer cells defined by • Ability to proliferate uncontrollably • Causes tumor formation • Angiogenesis feeds the tumor • Cancer cells increase secretion of angiogenesis activators and decrease angiogenesis inhibitors • Ability to spread through the body. • Invasion: cancer cells migrate into surrounding tissues, blood vessels • Metastasis: cancer cells spread through circulatory system to establish secondary tumors.

19. Normal versus tumor growth in skin • Cancer cells don’t necessarily divide faster than normal cells. • Normal: Rates of cell division and differentiation balanced. • Tumor: Balance of cell division and cell differentiation favors cell division.

20. Normal cells Regulated growth Dependent on GF’s Subject to cycle cycle control Anchorage-dependent Contact inhibited (density-dependent) Subject to apoptosis Flattened cells, normal nuclei, normal chromosome number Transformed (cancer) cells Uncontrolled growth Independent of GF’s Don’t obey checkpoints Anchorage-independent Loss of contact inhibition > pile up in culture Escape apoptosis pathways Rounded cells, large nuclei, abnormal chromosome number Normal and transformed cell properties