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Germ vs. Somatic cells

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					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….
                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.
    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.
Establishment of somatic cells
           Gastrulation generates the three
              classes of somatic cells
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Three classes of somatic cells
     • Ectoderm (outside)
     • Endoderm (inside)
     • Mesoderm (in between)
        – frequently called the 3 germ layers
   Establishment of the germline

• Two different ways in different organisms

       1) Preformation (segregation)

         2) Epigenesis (recreation)



               Extavour and Akam, Development 130:5896 (2003)
  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
     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.

        QuickTime™ an d a
TIFF (Uncompressed) decompressor       c) After first cell division all P-
  are need ed to see this p icture .   granules in Posterior cell.



                                       d) At 4-cell stage all P-granules
                                       are in Posterior P1 cell
       Germline formation in C. elegans




6 Founder Cells                           +98 die




                                      +14 die

Small posterior cell goes
       germline
                                                +1 dies
          Germline formation in C. elegans



8-cell


DAPI staining                           Antibodies to
of nuclei                               P-granules




24-cell
                   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)
  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.
Epigenesis of germline in mouse
                                           Developmental Cell 2: 537
                                           (2002) Zhao and Garbers,
                                           Male Germ Cell Specification
                                           and Differentiation


                                   Blue = embryonic
                                   white = extraembryonic




                   Embryonic   Embryonic   Embryonic
                   Ectoderm    Mesoderm    Endoderm
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
               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.
        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.
     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
     Stem Cell Biology
1)   Definition of stem cells




                     Generation of
                     more restricted
Self-renewal
                     progeny (lineage-
(expansion?)            commitment)
       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
              Types of stem cells
        1)    Embryonal Stem Cells (ES)
        2)    Embryonal Germ Cells (EG) embryonic
        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
adult   9)    Epidermal stem cell
        10)   Corneal stem cells
        11)   Mammary gland stem cells
        12)   Others…..
     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.
   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
 Properties of ES cells

• Pluripotent
  – Germ line and all somatic tissues, no
    extraembyronic tissues


• Immortal?



• Can be genetically modified
Generation of chimeric mice
        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)
   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.
More than one pluripotent embryo-
        derived stem cell



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Recommended reading
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                                                                                               2009




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     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…….
    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?
    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
Hematopoietic Stem Cells
 Hematopoietic Stem Cells

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
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"
   Properties of Hematopoietic
           Stem Cells
1) Thy1lo, 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.
Properties of Hematopoietic
        Stem Cells




              Weissman
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.
      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…..
                           Epigenetic landscape
                                                  (Waddington, 1957)




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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 Hochedlinger1 and Kathrin Plath2
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
      Comparison of stem cell
            features
ES, EG, EpiSc, iPSC          Adult stem
 • Pluripotent             • Multipotent or
 • Fast growing              unipotent
 • Easily cultured,        • Slow growing
   purified                • Difficult to
 • Genetically               culture, purify
   manipulable             • Hard to
 • Can be differentiated     manipulate
   in vitro                • Can often
                             differentiate
        For Thursday
Reprogramming of somatic cells
         into iPSCs
• Download papers and handouts
• Read papers carefully and be ready to
  discuss 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
  Thursday

				
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