Germ cells from ES cells 3

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					  GERM CELLS
 DERIVED FROM
EMBRYONIC STEM
     CELLS
PRESENTED BY CB ALLARD
     AND ANNA YU
            INTRODUCTION
1.   EMBRYONIC STEM CELLS

2.   PRIMORDIAL GERM CELLS

3.   NORMAL GERM CELL DEVELOPMENT

4.   OUTLINE OF EXPERIMENTS
1. EMBRYONIC STEM CELLS
   Overview:
     Typically derived from blastocyst in vitro
     Can be maintained indefinitely

     Pluripotency/Totipotency
         Capable of developing into many (pluripotent) or any (totipotent) cell types
         Oct-4 expression indicates pluripotency

         Embryoid bodies develop when LIF is removed
1. EMBRYONIC STEM CELLS
   Implications:
     Organ regeneration/transplants
     Studies of diseased tissue and mutation effects on development

     Unforeseen medical benefits

     Ethical concerns
           Experiments require eggs from donors
    2. PRIMORDIAL GERM CELLS




   Germ cells are derived from PGCs
   Differentiate from the proximal epiblast
   Detectable by staining for alkaline phosphatase
   Late gastrulation: PGCs migrate to gonads, then differentiate into egg or sperm
          2. PRIMORDIAL GERM CELL
   Migrate to genital ridge, which
    develops into gonad and induces
    PGCs to develop into germ cells
          3. NORMAL GERM CELL
              DEVELOPMENT
   Sex of the germ cell is determined by induction from the gonad,
    not from germ cell genes (recall sperm may carry X
    chromosome)
       Follicles: female somatic gonad cells which surround developing oocyte
        and synthesize estradiol
          3. NORMAL GERM CELL
              DEVELOPMENT
   Females: diploid germ cells arrest at meiosis prophase I until sexual
    maturity; undergo final meiotic divisions prior to fertilization


   Males: diploid germ cells arrest in G1 – enter meiosis 7-8 days after birth

                       Meiosis markers are indicators of
                           germ cell development
              3. NORMAL GERM CELL
                  DEVELOPMENT
   IMPRINTING
      Germ cells must be highly specialized, but also be capable of “starting
       over” at zygote stage
         A  female egg’s haploid genotype may find itself in a male sperm in the
           next generation.
     –Hence, alterations made during specialization are erasable in next
       generation
   IMPRINTING
       Normal development of embryo requires haploid genome from each parent, not just diploidy
       Genome “remembers” which parent it came from using methylation markers (recall sex
        chromosomes insufficient – an X genome can come from a male) which shut off certain
        genes
       Imprinting remains during development but must be erased in the germ cell line
                 4. OVERVIEW OF
                  EXPERIMENTS
   Allowed stem cells to differentiate into embryoid bodies (EBs)
   Detected and isolated PGC-like entities from EBs
   PGCs differentiated into gamete-like cells (oocytes and sperm)
   Various markers used to identify PGCs, oocytes, and sperm
 Visualizing germ line: expression of
             gcOct4-GFP
1.   Oct-4 gene expression specific to germ line and epiblast in EBs
2.   Deleted epiblast promoter region, leaving the germ cell
     promoter intact  only expressed in germ cell and not in
     epiblast
3.   Inserted GFP gene in place of Oct4 gene
4. Transfected ES cells with gcOct4-GFP and cloned
5. gcOct4-GFP only expressed in found germ cells
Derivation of embryonic GCs and
  male gametes from ES cells
      (Geijsen et al 2003)
   ES cells experimentally induced to differentiate into EBs
   Quantified gene expression and looked for germ line markers
     Isolated RNA from ES cells, EBs at different times of development,
      and testis (control)
     Amplified by RT-PCR
                   Gene expression markers
1. pluripotent ES cells (undifferentiated) and PGCs
         Oct4
         SSEA1 surface antigens
2. Mature germ cells
         stella
         fragilis (Fgls)
3. Expression in germ line and not in soma:
         Dazl              Rnh2
         Piwil2 Tdrd1
         Rnf17 Tex14
Undifferentiated embryonic stem cells and
                    primordial germ cells


                      Germ cell markers




                      Germ line markers
                           (not in soma)




                                  control
               Which genes expressed where?
1.   All marker genes expressed in undifferentiated ES cells
2.   From ES to EB - decreased expression of almost all marker genes
3.   Exception: rare population of cells expressed Oct-4 and SSEA1 as EBs
     developed
                           Oct-4+/SSEA1+ cells =
                   remainder of undifferentiated ES cells?
                    OR PRIMORDIAL GERM CELLS???
Rare population of Oct-4+/SSEA1+ cells: undifferentiated ES
                    cells or germ cells?
Retinoic acid differentiates between ES cells and germ cells:
1.   ES cells stop dividing and differentiate
2.   germ cells proliferate
Oct-4+/SSEA1+ cells from EBs proliferated 
            most likely PGCs
     Further evidence that Oct-4+/SSEA1+ cells were
                          PGCs
      Stained for alkaline phosphatase in SSEA1+ EBs cells
1.     Small population of SSEA1+ showed positive staining
2.     Positive stained cells surrounded by moving cells resembling
       migratory PGCs
             Erasure of epigenic imprints?
   PGCs are the only cells that show erasure of imprints
   Igf2r has region DMR2 that is hypermethylated only on the
    maternal allele
   Hypothesis: if the cells in question are PGCs, then methylation
    should not be detected at DMR2
            Erasure of epigenic imprinting?
1.   Leukemia inhibitory factor, stem cell factor, and basic
     fibroblast growth factor placed in culture (support development
     of embryonic germ cells)
2.   Digested EB genome with PvuII to run through gel
3.   Put in Mlu1 (methylation sensitive enzyme) – does not digest
     methylated genes
By day 10 all imprinting signals erased in Igf2r DMR2
                        region
                    Quick summary:
1.    Rare population of cells had Oct4 and SSEA1 expression after ES cell
      differentiation
2.    Rare population of cells proliferated in RA
3.    Rare population of cells stained positive for alkaline phosphatase
4.    Rare population of cells showed epigenic erasure
     RARE POPULATION OF CELLS DISPLAY IN VIVO PRIMORDIAL
                       GERM CELL CHARACTERISTICS
     Are the PGCs in a defined region within the embryoid
                            body?
      Immunohistochemical analysis of 7 day old EBs
1.     CD41 labels haematopoietic cells
2.     SSEA1 labels germ cells

       Both cell populations found very close together, as in vivo
     Do PGCs differentiate into “normal” gametes?

1.   Detected Sry in day 5 of EB development
2.   Day 11, strong upregulation of acrosin and haprin
3.   No expression of ZP1, 2, or 3 (female gametogenisis genes)
PGCs showed
characteristic gene
expression of sperm
development
      Do these male germ cells undergo proper
                     meiosis?
   FE-J124 – antibody that binds male meiotic germ cells

   Hoechst 33342 – fluorescent dye that binds to DNA for
    quantification purposes
Inefficient meiosis of EB derived male germ cells

   EB derived FE-J1+ germ cells had
    lower proportion haploidy (36%)
    than cells from adult mice testes
    (68%)
Are EB derived haploid cells biologically competent as sperm
                            cells?
   Isolated FE-J1+/Oct-4+ cells from day 20 EBs
   Intracytoplasmic injections into oocytes at two independent labs -
    same results
   one in five developed into blastocysts
     Derivation of Oocytes from Mouse
           Embryonic Stem Cells
            (Hubner et al 2003):
      allow ES cells to differentiate
     Identify cells with oocyte characteristics
1.    gcOct4-GFP and c-kit expression marks early germ cells
      (gcOct4-GFP expressions = Oct4 expression)
2.    Vasa – post-migratory germ cells
3.    SCP3 and DMC1 – meiosis specific markers
Separated into four populations of cells based on expression of
                     gcOct4-GFP and ckit
            Formation of follicular structures:
   cell aggregates collected by centrifugation and then cultured
   some looked like early ovarian follicles
   about 20% of those follicles produced oocytes larger than 40um
     Do follicle-like structures produce estradiol?
   Natural follicles make estradiol
   Hypothesis: if detect estradiol in culture, then follicle like
    structures are probably follicles
   Result: found estradiol by day 12, peaked at day 20
                Characterization of Oocytes
   Day 26, detected oocyte-like cells released from somatic cells
   Hypothesis: if oocyte specific gene markers (ZP1, ZP2, ZP3, Fig
    alpha, GDF-9) are found in the cells released, they may be
    oocytes
   Results: all expressed except ZP1, which may explain weak zona
    prone to detaching
                      Meiosis detection?

   After 16 days, expressed proteins typical of meiosis
   SCP3/COR1 staining – in stage before leptotene (first phase of
    prophase I)
Surprise: blastocyst like structure derived from ES
                         cells
   follicular outgrowths similar to oocytes that have been
    parthenogenetically activated
   May be artifact of lab techniques
   None survived to birth after implantation
    Summary of the experiments
   ES cells, somatic cells, germ cells, gametes all have different RNA
    and protein markers
   These differences make it possible to distinguish between
    different cell types
     Summary of sperm derivation
1.    Oct-4+/SSEA1+ cells proliferated in retinoic acid; stained positive for
      alkaline phosphatase  most likely PGCs (rather than undifferentiated
      ES cells)
2.    Showed erasure of epigenic imprints, similar to in vivo germ cells
3.    PGCs showed characteristic gene expression of male gamete
      development (Sry, Gcnf, acrosin, haprin)
4.    Inefficient meiosis – about half the amount of cells meiotic compared to
      testis cells
5.    Fertilized donor eggs – 20% developed into blastocysts
 Summary of oocyte derivation
1.   Oct4, c-kit, Vasa detection implied germ cell development
2.   Formation of follicle-like structures, 20% developing into 40um oocytes
3.   Estradiol production by follicles
4.   oocyte-like cells released from somatic cells; expressed oocyte specific
     gene markers (ZP2, ZP3, Fig alpha, GDF-9)
5.   Meiosis detection
6.   Blastocyst like structures arose without fertilization or parthogenic
     activation
               DISCUSSION
1.   POTENTIAL MEDICAL BENEFITS

2.   POTENTIAL RESEARCH BENEFITS

3.   ETHICAL IMPLICATIONS

4.   LIMITATIONS/FURTHER RESEARCH
         1. POTENTIAL MEDICAL
               BENEFITS
   Fertility treatment
      Understand causes of infertility
      Use synthetic sperm to treat male infertility

   Germ-cell tumors
      Understanding the causes
      Designing treatment

   Recipients for nuclear transplants, gene therapy, etc.
              1. POTENTIAL MEDICAL
                    BENEFITS
   Nuclear transplants into
    stem cell-derived oocytes
        2. POTENTIAL RESEARCH
               BENEFITS
   Understanding imprinting and human germ cell development
   A limitless supply of eggs could be derived from a single line of
    ES cells
       Use of eggs to produce diseased tissue for study
        2. POTENTIAL RESEARCH
               BENEFITS
   Study of sex chromosomes’ effects on development:
     Oocytes were derived from male stem cells (diploid – XY)
     Study effects on development of a YY zygote

     Potential to study meiosis in a YY oocyte
           2. POTENTIAL RESEARCH
                  BENEFITS
   TRISTEM
     Company in London, UK
     Claims to have developed technique to successfully transform white blood cells
      into stem cells
     Proof of pluripotency but not yet of totipotency

     If true, potential to derive all types of cells of an individual’s own genotype –
      potential for fertility treatment, organ regeneration (without rejection), cancer
      therapy, etc.
     Bypasses ethical concerns too
3. ETHICAL IMPLICACTIONS
   Stem cell research uses human tissues, but:
      1.   Donor consent is given

      2.   only for acceptable purposes – what is acceptable though?

      3.   Avoid inappropriate commercialization
  3. ETHICAL IMPLICATIONS
4. Embryo having the potential to become an autonomous being - person has
     a right to life
        Can a blastocyst be considered as a person?
        Is a cell worth the same respect as an embryo?
5. Medical advancement worth the consequences of cloning?
6. human cloning wasteful of embryos and fetuses (cloned embryos have had
     poor success of surviving to adulthood)
  3. ETHICAL IMPLICATIONS
7. autonomy (right of self government)

8. Sex selection is legal in Canada and USA for non-medical reasons

9. Designer babies
                       4. Limitations:
1. In vitro vs. in vivo
        inefficient meiosis in sperm
        parthenotes formed by uninduced/unfertilized eggs
        nuclear transfer experiments in oocytes may be questionable, given their weak
         membrane
2. Future studies
        oocytes – whether they show erasure of epigenic imprints
        Can ES cell derived germ cells fertilize each other and will it grow into normal
         embryo?
3. Legislature? Implications of doing such research (illegal cloning? Designer
     babies?)

				
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