GERM CELLS DERIVED FROM EMBRYONIC STEM CELLS

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GERM CELLS DERIVED FROM EMBRYONIC STEM CELLS

• PRESENTED BY CB ALLARD AND ANNA YU

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INTRODUCTION

• EMBRYONIC STEM CELLS
• PRIMORDIAL GERM CELLS
• NORMAL GERM CELL DEVELOPMENT
• OUTLINE OF EXPERIMENTS

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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

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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

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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

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2. PRIMORDIAL GERM CELL

• Migrate to genital ridge, which develops into
  gonad and induces PGCs to develop into germ cells

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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

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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

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3. NORMAL GERM CELL DEVELOPMENT

• IMPRINTING
• Germ cells must be highly specialized, but also
  be capable of starting over at zygote stage
• A female eggs haploid genotype may find itself
  in a male sperm in the next generation.
• Hence, alterations made during specialization
  are erasable in next generation

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• 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

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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

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Visualizing germ line expression of gcOct4-GFP

• Oct-4 gene expression specific to germ line and
  epiblast in EBs
• Deleted epiblast promoter region, leaving the
  germ cell promoter intact ? only expressed in
  germ cell and not in epiblast
• Inserted GFP gene in place of Oct4 gene

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• 4. Transfected ES cells with gcOct4-GFP and
  cloned
• 5. gcOct4-GFP only expressed in found germ cells

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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

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Gene expression markers

• 1. pluripotent ES cells (undifferentiated) and
  PGCs
• Oct4SSEA1 surface antigens
• 2. Mature germ cells stella fragilis (Fgls)
• 3. Expression in germ line and not in soma
• Dazl Rnh2
• Piwil2 Tdrd1
• Rnf17 Tex14

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Undifferentiated embryonic stem cells and
primordial germ cells
Germ cell markers
Germ line markers (not in soma)
control
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Which genes expressed where?

• All marker genes expressed in undifferentiated ES
  cells
• From ES to EB - decreased expression of almost
  all marker genes
• 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???

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Rare population of Oct-4/SSEA1 cells
undifferentiated ES cells or germ cells?

• Retinoic acid differentiates between ES cells and
  germ cells
• ES cells stop dividing and differentiate
• germ cells proliferate

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Oct-4/SSEA1 cells from EBs proliferated ? most
likely PGCs
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Further evidence that Oct-4/SSEA1 cells were
PGCs

• Stained for alkaline phosphatase in SSEA1 EBs
  cells
• Small population of SSEA1 showed positive
  staining
• Positive stained cells surrounded by moving cells
  resembling migratory PGCs

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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

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Erasure of epigenic imprinting?

• Leukemia inhibitory factor, stem cell factor, and
  basic fibroblast growth factor placed in culture
  (support development of embryonic germ cells)
• Digested EB genome with PvuII to run through gel
• Put in Mlu1 (methylation sensitive enzyme) does
  not digest methylated genes

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By day 10 all imprinting signals erased in Igf2r
DMR2 region
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Quick summary

• Rare population of cells had Oct4 and SSEA1
  expression after ES cell differentiation
• Rare population of cells proliferated in RA
• Rare population of cells stained positive for
  alkaline phosphatase
• Rare population of cells showed epigenic erasure
• RARE POPULATION OF CELLS DISPLAY IN VIVO
  PRIMORDIAL GERM CELL CHARACTERISTICS

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Are the PGCs in a defined region within the
embryoid body?

• Immunohistochemical analysis of 7 day old EBs
• CD41 labels haematopoietic cells
• SSEA1 labels germ cells
• Both cell populations found very close together,
  as in vivo

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Do PGCs differentiate into normal gametes?

• Detected Sry in day 5 of EB development
• Day 11, strong upregulation of acrosin and haprin
• No expression of ZP1, 2, or 3 (female
  gametogenisis genes)

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PGCs showed characteristic gene expression of
sperm development
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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

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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)

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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
•  

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Derivation of Oocytes from Mouse Embryonic Stem
Cells(Hubner et al 2003)

•  allow ES cells to differentiate
• Identify cells with oocyte characteristics
• gcOct4-GFP and c-kit expression marks early germ
  cells (gcOct4-GFP expressions Oct4 expression)
• Vasa post-migratory germ cells
• SCP3 and DMC1 meiosis specific markers

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Separated into four populations of cells based on
expression of gcOct4-GFP and ckit
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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

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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

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Characterization of Oocytes

• Day 26, detected oocyte-like cells released from
  somatic cells

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• 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

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Meiosis detection?

• After 16 days, expressed proteins typical of
  meiosis
• SCP3/COR1 staining in stage before leptotene
  (first phase of prophase I)

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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

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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

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Summary of sperm derivation

• Oct-4/SSEA1 cells proliferated in retinoic
  acid stained positive for alkaline phosphatase ?
  most likely PGCs (rather than undifferentiated ES
  cells)
• Showed erasure of epigenic imprints, similar to
  in vivo germ cells
• PGCs showed characteristic gene expression of
  male gamete development (Sry, Gcnf, acrosin,
  haprin)
• Inefficient meiosis about half the amount of
  cells meiotic compared to testis cells
• Fertilized donor eggs 20 developed into
  blastocysts

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Summary of oocyte derivation

• Oct4, c-kit, Vasa detection implied germ cell
  development
• Formation of follicle-like structures, 20
  developing into 40um oocytes
• Estradiol production by follicles
• oocyte-like cells released from somatic cells
  expressed oocyte specific gene markers (ZP2, ZP3,
  Fig alpha, GDF-9)
• Meiosis detection
• Blastocyst like structures arose without
  fertilization or parthogenic activation

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DISCUSSION

• POTENTIAL MEDICAL BENEFITS
• POTENTIAL RESEARCH BENEFITS
• ETHICAL IMPLICATIONS
• LIMITATIONS/FURTHER RESEARCH

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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.

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1. POTENTIAL MEDICAL BENEFITS

• Nuclear transplants into stem cell-derived oocytes

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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

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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

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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 individuals own genotype potential for
  fertility treatment, organ regeneration (without
  rejection), cancer therapy, etc.
• Bypasses ethical concerns too

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3. ETHICAL IMPLICACTIONS

• Stem cell research uses human tissues, but
• Donor consent is given
• only for acceptable purposes what is acceptable
  though?
• Avoid inappropriate commercialization

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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)

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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

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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?)