Stem Cells: Hype and Promise

1. Stem Cells: Hype and Promise What they are, how they are used and can they ever provide clinical benefits?

2. Today’s Agenda • Historical perspectives • Hematopoietic (blood forming) stem cells • Embryonic (totipotent) stem (ES) cells • Cancer stem cells • Possible futures

3. Definitions • Pluripotent stem cells: cells capable of limited differentiation potential, i.e. blood forming cells • Totipotent stem cells: cells capable of giving rise to an entire animal, i.e. embryonic stem cells • Cancer stem cells: mutated pluripotent(?) stem cells that give rise to tumors • Cloning: The derivation of an exact piece of DNA, cell or entire organism from a pre-existing entity

4. Early Clues in the “pre-cellular” world (18th century) • Regeneration in hydra-cut the organism (a simple invertebrate) in two and get 2 new organisms • Regeneration in echinoderms-cut off a starfish (a complex invertebrate) leg and watch as a new one grows • Regeneration in amphibians-cut off an amphibian (a vertebrate) leg or tail and watch a new one grow • The conclusion is that there is something (cells) in the adult capable of forming new and complex organs

5. 20th century cell biology: mouse teratomas • Teratomas are “germ cell” (sperm/oocyte) tumors that give rise to a variety of tissues including teeth and hair! • The stem cells of the teratoma are cancerous, while the differentiated cells are not • “Transplantation” of cancerous teratoma stem cells into normal embryos gives rise to completely “normal” adults. • These results suggested that stem cells could be studied and used to give rise to entire animals • These results were also the first to suggest that animals could be “cloned”.

6. Immature Teratoma

7. Hematopoietic (blood-forming) stem cells-the “pluripotent cell” • Early microscopy data suggested that skin and gut cells turn over rapidly and, therefore, they must be replenished • Even more striking was the turnover of blood cells, particularly white blood cells (leukocytes) • Billions of these cells must be replaced daily, and the source of these new cells was clearly the bone marrow • The effects of radiation on the bone marrow of A-bomb victims led scientists to show that X-rays were lethal mainly because of their effects on blood cell formation • This led to an explosion of data in murine systems on the nature of the blood-forming stem cell

8. Mice are very good for some things, like studying blood stem cells • Anemic mice could be “rescued” by transplantation of embryonic blood cells • Transplantation of bone marrow cells into gamma-irradiated mice resulted in colonies of mixed blood cells growing in spleens-the first evidence for a blood stem cell • The hematopoietic stem cell is extremely rare (<.01%) • 25 years and countless irradiated mice were required to isolate a cell that, when injected into a lethally irradiated animal, could replenish the bone marrow and mature blood • This cell is the pluripotent hematopoietic stem cell • Bone marrow transplants are now routine clinical procedures

9. Deterministic model of hematopoiesis

10. Tissue culture changes everything-the “totipotent cell” • Tissue culture is the ability to grow cells in plastic dishes using nutrient medium • This allows for the isolation of large numbers of cells for further study • While teratoma stem cells could be grown in culture, there was a nagging suspicion that they were abnormal • Therefore, a search was done to isolate normal counterparts of these tumor stem cells from embryos • This resulted in the isolation and growth in culture of embryonic stem (ES) cells, derived from the “inner cell mass” of the embryo

11. Derivation of murine ES cells • Inner cell mass (ICM) gives rise to the embryo • ICM is microsurgically removed • ICM is placed in tissue culture with “feeder cells” and grows • A single ES cell from the ICM can make an entire mouse • It is, therefore, “toptipotent”

12. An entire mouse can be made from a single ES cell • ES cells can be grown in culture permanently • The cell is plucked out of culture and injected into the ICM of a different strain of mice • The injected blastocysts is transplanted into an adult uterus • Offspring derived from the transplanted cell have different colored coats • They are genetically identical clones of the ES cell • The same thing can be done with any species, including us

13. Microsurgery on an egg

14. Mutant mice can be made from genetically manipulated ES cells

15. Cancer stem cells • Previous work suggested that teratomas contained a tumor stem cell • Blood tumors (leukemias) also were found to contain a stem cell • A search was undertaken in solid tumors (brain and breast) for a stem cell that gave rise to the tumor

16. Breast cancer stem cells give rise to different cell types Stochastic model Cancer stem cell model CSC CSC CSC CSC Different clonogenic cells within the tumor give rise to phenotypically similar cells in new tumors. Cancer stem cells give rise to phenotypically diverse cells that recapitulate the complexity of the original tumor.

17. Isolation of cancer stem cells Flow Cytometry

18. Tumor formation by human breast cancer cells B38.1+CD44+ CD24- injection B38.1+CD44+ CD24+ injection

19. Therapeutic Implications of Cancer Stem Cells Therapy against an oncogenic mutation that is expressed by cancer stem cells The tumor is eliminated Therapy against an oncogenic mutation that is not expressed by cancer stem cells The tumor inititially shrinks- but The self-renewing cancer stem cell regenerates the tumor

20. Possible futures • Production of genetically desirable organisms • Production of new tissues in culture • Cloning of humans

21. Production of genetically desirable organisms • This has already been done with plants-so called “GM” crops-these modified stem cells (seeds) can be made resistant to a diversity of pathogens and herbicides • Using ES cells, any gene can be easily mutated or overexpressed • The modified ES cells can subsequently be used to give rise to a whole animal containing the mutated/overexpressed gene • This will likely be useful in food production as well as animals producing pharmaceuticals

22. Rapid engineering of genetically desirable traits into animals

23. Production of new tissues in culture • ES cells can be used to form a diversity of differentiated cells in different culture conditions culture • Blood, cardiac myocytes, neurons etc. have all been differentiated from totipotent ES cells • The problem is that there are insufficient numbers of these cells and, with the exception of blood, they are hard to integrate into existing organs • While highly hyped, this procedure is a long way from clinical applicability

24. Tissue replacement with culture-differentiated stem cells

25. Why would anyone want to be cloned? • Some people are psychotic • It would allow for possibly greatly enhanced lifespan • Because the clone would be genetically identical, organs and blood could be transplanted as we age • This would take care of many of the diseases that kill us • There are obviously ethical problems with this, for example, the clone, which is another human, would need to be dispatched to get the matched tissues (except for some cases, like blood, one kidney, etc.)

26. Could any of us be “cloned”? • Amazingly, yes (with caveats) • Early frog experiments showed that a nucleus from a completely differentiated cell could give rise to an entire frog • Decades later, the same experiment was repeated in mammals (“dolly”) • Most animals born this way have many physiological problems

27. Nuclear Cloning

28. The bottom line on mammalian cloning

29. Further Reading (Amazon) • The Proteus Effect: Stem cells and Their Promise for Medicine, Ann Parsons • Human Embryonic Stem Cells: an Introduction to the Science and Therapeutic Potential, Ann Kiessling and Scott Anderson • Stem Cells: Scientific Progress and Future Research Directions: National Institutes of Health

30. Enjoyment Reading • Einstein: 1905 the Standard of Greatness, John Rigden • Incompleteness: the Proof and Paradox of Kurt Goedel, Rebecca Goldstein • The Great Mortality: an Intimate History of the Black Death, the Most Devastating Plague of All Time, John Kelly