1. Somatic, Germ and Stem cells
Distinctions between somatic and germ cells.
Establishment of somatic cell types.
Establishment of the germ line.
Fundamental differences in germ cell establishment between organisms.
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. Germ vs. Somatic cells
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.

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
Small posterior cell goes
germline
+1 dies

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

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
Blue = embryonic
white = extraembryonic
Embryonic
Ectoderm
Embryonic
Mesoderm
Embryonic
Endoderm

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

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
Shamblott et al. PNAS 95:13726 (1998)
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.

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

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

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

21. Types of stem cells embryonic adult Embryonal Stem Cells (ES)
Embryonal Germ Cells (EG)
Epiblast stem cell (EpiSC)
Induced Pluripotent Stem Cells (iPSCs)
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
Others…..
embryonic
adult

22. Embryonal Stem (ES) cells
Derived from inner cell mass of blastocysts
Nature (5965): 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 Immortal?
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 Immortal?
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. Hematopoietic Stem Cells
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
Finding appropriate markers.
lineage markers
Developing better assays.
competition assay vs. rescue
Cell sorting protocols.
FACS
Better understanding of "reconstitution".
"Short-term"vs. "Long-term"

37. Properties of Hematopoietic Stem Cells
Thy1lo, c-kit+, Sca-1+.
Negative for B220, Mac-1, Gr-1, CD3, 4, 8, Ter119.
Are present as 0.007% of bone marrow.
Can do long-term reconstitution of all blood cell types with single (or small numbers) of cells.
Are resistant to killing with S-phase drugs or labeling with BrdU.
Last several lifetimes.

38. Traditional schematic of hematopoietic development indicating intermediates in the hierarchy of hematopoietic differentiation.
Schematic of hematopoietic development indicating intermediates in the hierarchy of hematopoietic differentiation. Surface markers used for isolation are indicated at left for human (top) and mouse (bottom) for each stem and progenitor cell. HSC indicates long-term reconstituting, self-renewing; MPP, multipotent progenitors with limited self-renewal leading to transient but multilineage reconstitution; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; BLP, B lymphocyte protenitor; ProT, T-cell progenitor; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/erythroid progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor. Adapted from Figure 1 in Bryder D, Rossi DJ, Weissman IL. Hematopoietic stem cells: The paradigmatic tissue specific stem cell. Am J Pathol. 2006;169: , with permission from the American Society for Investigative Pathology.
©2008 by American Society of Hematology
Weissman I L , and Shizuru J A Blood 2008;112:

39. A new model of Hematopoiesis from colony forming assays
Notta et al.

40. Models of Hematopoiesis from RNA-seq
Mercier and Scadden Cell 2015

41. Most recent model of Hematopoiesis from RNA-seq
Dynamic
Instability
"Genetic perturbations and chromatin immuno-precipitation followed by sequencing revealed Irf8 and Gfi1 as key components of counteracting myeloid-gene-regulatory networks. Combined loss of these two determinants ‘trapped’ the metastable intermediate. We propose that mixed-lineage states are obligatory during cell fate specification, manifest differing frequencies because of their dynamic instability and are dictated by counteracting gene regulatory networks."
Olsson et al.

42. Lessons from the search for the Hematopoietic Stem Cell
Develop quantitative assays.
Find appropriate markers.
Develop in vitro assays, culture systems.
Define lineage of cells of interest.

43. Types of stem cells http://stemcells.nih.gov/info/glossary.asp
Embryonal Stem Cells (ES)
Embryonal germ cells (EGC)
Epiblast Stem Cells (EpiSC)
Induced Pluripotent Stem Cells (iPSC)
Hematopoietic Stem Cells (HSC)*
Intestinal "crypt" stem cell
Olfactory neuron stem cell
Cardiac and skeletal muscle stem cells
Neuronal/glial stem cell
Epidermal stem cell
Corneal stem cells
Mammary gland stem cells
Others…..

44. 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, (2009) doi: /dev
Epigenetic reprogramming and induced pluripotency
Konrad Hochedlinger1 and Kathrin Plath2

45. 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

46. Comparison of stem cell features
ES, EG, EpiSc, iPSC
Adult stem
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

47. 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 Figure Facts sheets and them before class on Thursday
We'll discuss how this work has progressed at the end of class on Thursday.