1. Somatic, Germ and Stem cells 1)Distinctions between somatic and germ cells. 2)Establishment of somatic cell types. 3)Establishment of the germ line. 4)Fundamental differences in germ cell establishment between organisms. 5)Where stem cells fit in….

2. Cell types Two basic classes of cells in animals –somatic cells –germ cells Somatic cells - almost everything we see and use, skin, muscle, blood, etc. Germ cells - the cells that are responsible for reproduction, propagation of the species. Stem cell - neither fish nor fowl. Can in some cases do both.

3. 1) These two classes of cells are established in different ways during development. 2) There are two fundamentally different processes by which germ cells are generated in different organisms. Germ vs. Somatic cells

4. Establishment of somatic cells

5. Gastrulation generates the three classes of somatic cells

6. Three classes of somatic cells Ectoderm (outside) Endoderm (inside) Mesoderm (in between) –frequently called the 3 germ layers

7. Establishment of the germline Two different ways in different organisms 1) Preformation (segregation) 2) Epigenesis (recreation) Extavour and Akam, Development 130:5896 (2003)

8. Establishment of the germline Preformation –Germ cells determinants (germ plasm) are set aside before or at fertilization and remain segregated throughout the lifespan of the organism. C. elegans Drosophila

9. Germline formation in C. elegans In worms, the point of sperm entry defines the posterior pole of the egg. a) A fertilized egg with evenly distributed P-granules b) At pronuclear migration and fusion, P-granules move to the posterior, where the sperm nucleus was located. c) After first cell division all P- granules in Posterior cell. d) At 4-cell stage all P-granules are in Posterior P1 cell

10. Germline formation in C. elegans 6 Founder Cells +98 die +14 die +1 dies Small posterior cell goes germline

11. Germline formation in C. elegans 8-cell 24-cell Antibodies to P-granules DAPI staining of nuclei

12. Preformation C. elegans, Drosophila and many other model organisms. Some amphibians, birds. Segregation of mRNAs and proteins required for germ cell formation and function (germ plasm). –bruno (RNP-type binding protein, regulates translation) –gld-1 (RNA binding protein) –mex-1 (zinc-finger DNA-binding protein) –vasa (DEAD-box RNA helicase, interacts with eIF5b) –germ cell less (transc. repressor, binds E2F) –cycB (B-type cyclin, interacts with CDK1, ubiquitous) –tudor (Tudor domain containing, part of germ granules)

13. Epigenesis of germline in mammals Major distinction, no preexisting germ plasm in oocytes. Primordial germ cells are generated during development. Seen in all mammals (so far), many other species. Shared feature with Preformation: germ cells are "set aside" before other cell types.

14. Epigenesis of germline in mouse Developmental Cell 2: 537 (2002) Zhao and Garbers, Male Germ Cell Specification and Differentiation Embryonic Ectoderm Embryonic Mesoderm Embryonic Endoderm Blue = embryonic white = extraembryonic

15. Epigenesis of germline in mouse BioEssays 27.2, Matsui and Okamura, Mechanisms of germ-cell specification in mouse embryos. 2005 BMP-4, -8b BMP receptor

16. Primordial germ cells Generated by epiblast. Separate from the 3 somatic cell layers. Migrate to gonadal ridge. (Molyneaux et al. Dev. Biol. 240:488 (2001). Populate ridge to form germ cells of gonad. In vitro, PGCs can differentiate into Embryonal Germ Cells (EGC). (Donovan et al. Cell 44:831 (1986), Matsui et al. Cell 70:841 (1992)). This differentiation is similar to the "reprograming" of somatic nuclei seen in Oocytes and ES cells. EGCs are one of only a few pluripotent stem cells currently known. Can contribute to all cell types in embryos.

17. Human Embryonal Germ Cells Human EGCs Mouse ES cells Cells were grown in DMEM supplemented with 15% fetal bovine serum, 0.1mM nonessential amino acids), 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml of penicillin, 100 ug/ml of streptomycin, 1,000 units/ml of human recombinant leukemia inhibitory factor (hrLIF), 1 ng/ml of human recombinant basic fibroblast growth factor (hrbFGF), and 10 mM forskolin. Shamblott et al. PNAS 95:13726 (1998)

18. Stem Cell Biology 1) Definition of stem cells 2) Types of stem cells 3) Derivation of stem cells 4) Functions of stem cells 5) Promise of stem cells

19. Stem Cell Biology 1) Definition of stem cells Self-renewal (expansion?) Generation of more restricted progeny (lineage- commitment)

20. Types of stem cells 1)What defines a type of stem cell? A)Where and how it was derived? B)What is its POTENTIAL? 2)Potentiality How many types of cells can the stem cell generate? Totipotent -> pluripotent -> unipotent

21. Types of stem cells 1)Embryonal Stem Cells (ES) 2)Embryonal Germ Cells (EG) 3)Epiblast stem cell (EpiSC) 4)Induced Pluripotent Stem Cells (iPSCs) 5)Hematopoietic Stem Cells (HSC) 6)Intestinal "crypt" stem cell 7)Cardiac and skeletal muscle stem cells 8)Neuronal/glial stem cell 9)Epidermal stem cell 10)Corneal stem cells 11)Mammary gland stem cells 12)Others….. adult embryonic

22. Embryonal Stem (ES) cells Derived from inner cell mass of blastocysts Nature. 1984 309(5965):255-6. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Bradley A, Evans M, Kaufman MH, Robertson E.

23. Embryonal Stem (ES) cells Normally cells go on to form embryo of mouse –Ectoderm, mesoderm, endoderm (3 germ layers) –Germ cells, germline cells In culture, cells can be manipulated and used to create novel mice Name Embryonic Stem Cell coined by Gail Martin.

24. Properties of ES cells Pluripotent –Germ line and all somatic tissues, no extraembyronic tissues Immortal? Can be genetically modified The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

25. Generation of chimeric mice

26. Properties of ES cells Pluripotent –Germ line and all somatic tissues Can differentiate in vitro into many cell types. Immortal? Can be genetically modified Genes essential for ESC pluripotency –Oct4 Niwa et al. Mol. Cell Biol. 22 (2000) –Nanog Mitsui et al. Cell 113 (2003) –Sox2 Avilion et al. Genes Dev. 17 (2002) –FoxD3 Hannah et al. Genes Dev. 16 (2002) –Stat3 signaling important (LIF)

27. Questions about ES cells How do they maintain pluripotency? How can we consistantly differentiate them in vitro? How similar are human ES cells to mouse? Can they (or something like them) be generated without using embryos (human)? Answered in 2006. All fundamental questions about the differentiated state.

28. More than one pluripotent embryo- derived stem cell ?? ??

29. Thursday reading 2009

30. Adult stem cells Hematopoietic Stem Cells (HSC) Intestinal "crypt" stem cell Cardiac and skeletal muscle stem cells Neuronal/glial stem cell Epidermal stem cell Corneal stem cells Mammary gland stem cells Embryonal germ cells Others…….

31. Hematopoietic Stem Cells McCulloch A younger me

32. Hematopoietic Stem Cells Discovered in 1961 (Till and McCulloch, Rad. Res. 14:213 (1961)) Rescue of lethal irradiation of mice –950 rads kill 100% of mice (3-30 days). –Mice die from anemia. –Injection of bone marrow from unirradiated mice rescues the irradiated mice. –How to quantify, identify and isolate rescuing cells?

33. Hematopoietic Stem Cells Rescue of lethal irradiation of mice –Irradiate recipient mice –Injection of bone marrow from non-irradiated mice –Looked at hematopoietic organs of recipients

34. Hematopoietic Stem Cells

35. 1) First quantification of "stem" cells. 2) Demonstration of different differentiated cell types from one cell. 3) Started race to purify HSC (1961). Essentially completed in 1991

36. Hematopoietic Stem Cells 1) Finding appropriate markers. lineage markers 2) Developing better assays. competition assay vs. rescue 3) Cell sorting protocols. FACS 4) Better understanding of "reconstitution". "Short-term"vs. "Long-term"

37. Properties of Hematopoietic Stem Cells 1) Thy1 lo, c-kit +, Sca-1 +. 2) Negative for B220, Mac-1, Gr-1, CD3, 4, 8, Ter119. 3) Are present as 0.007% of bone marrow. 4) Can do long-term reconstitution of all blood cell types with single (or small numbers) of cells. 5) Are resistant to killing with S-phase drugs or labeling with BrdU. 6) Last several lifetimes.

38. Properties of Hematopoietic Stem Cells Weissman

39. Lessons from the search for the Hematopoietic Stem Cell 1) Develop quantitative assays. 2) Find appropriate markers. 3) Develop in vitro assays, culture systems. 4) Define lineage of cells of interest.

40. Types of stem cells 1)Embryonal Stem Cells (ES) 2)Embryonal germ cells (EGC) 3)Epiblast Stem Cells (EpiSC) 4)Induced Pluripotent Stem Cells (iPSC) 5)Hematopoietic Stem Cells (HSC)* 6)Intestinal "crypt" stem cell 7)Olfactory neuron stem cell 8)Cardiac and skeletal muscle stem cells 9)Neuronal/glial stem cell 10)Epidermal stem cell 11)Corneal stem cells 12)Mammary gland stem cells 13)Others….. http://stemcells.nih.gov/info/glossary.asp

41. Epigenetic landscape (Waddington, 1957) Fig. 1. The developmental potential and epigenetic states of cells at different stages of development. A modification of C. H. Waddington’s epigenetic landscape model, showing cell populations with different developmental potentials (left) and their respective epigenetic states (right). Developmental restrictions can be illustrated as marbles rolling down a landscape into one of several valleys (cell fates). Colored marbles correspond to different differentiation states (purple, totipotent; blue, pluripotent; red, multipotent; green, unipotent). Examples of reprogramming processes are shown by dashed arrows. Development 136, 509-523 (2009) doi:10.1242/dev.020867 Epigenetic reprogramming and induced pluripotency Konrad Hochedlinger 1 and Kathrin Plath 2

42. Promise of stem cells Diabetes Spinal cord injuries Stroke Heart Disease Neurodegenerative diseases Multiple Sclerosis Arthritis Asthma, Emphasema Spina Bifida Cerebral Palsey Cancer Kidney failure Amputations

43. Comparison of stem cell features Pluripotent Fast growing Easily cultured, purified Genetically manipulable Can be differentiated in vitro Multipotent or unipotent Slow growing Difficult to culture, purify Hard to manipulate Can often differentiate ES, EG, EpiSc, iPSCAdult stem

44. For Thursday Reprogramming of somatic cells into iPSCs Download papers and handouts Read papers carefully and be ready to discuss the original paper in class Fill out handout and turn in at beginning of class We'll discuss how this work has progressed at the end of class on Friday.