Embryoid%20Bodies%20From%20Embryonic%20Stem%20Cells%20In-vitro

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Embryoid Bodies From Embryonic Stem Cells In-vitro

• Robert Christensen
• Biology Department
• Eastern Connecticut State University

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EndodermSpecific Gene Expression in Embryonic
StemCells Differentiated To Embryoid Bodies

• Experimental Cell Research 229 (1999) pp 27-34

Koichiro Abe, Hitoshi Niwa, Katsuro Iwase, Masaki
Takiguchi, Masataka Mori, Shin Ichi Abe, Kuniya
Abe, and Ken- Ichi Yamamura
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Introduction

• Cell lineages arise during development

• ES cells have full developmental potential

• EBs from ES cells

• Ectodermal tissues
• Mesodermal tissues
• Endodermal tissues

• EBs consist of

• EBs resemble the embryo of the egg- cylinder
  stage

• Neuronal cells
• Cardiac muscle cells
• Hematopoietic cells
• Yolk sac cells

• Later stages EBs are composed of

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• Past studies showed specific changes in
  expression of endoderm marker genes
  during EB development and suggested that EB
  formation could be considered an in-vitro model
  for endoderm differentiation.

• This paper extended their analysis and
  systematically characterized temporal expression
  patterns of endoderm marker genes during EB
  formation.

• RNA blot
• Reverse transcription polymerase chain reaction
  (RT-PCR)
• In-situ hybridization

• The nature of endoderm differentiation in-vitro
  is discussed in relation to normal embryonic and
  extraembryonic development in-vivo.

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Materials and Methods

• Cell cultures

• ES cell line, D3, cultured

• Incubated 3 days in Dulbeccos modified eagles
  medium (DMEM)

• 15 fetal calf serum
• 0.1 mM 2-mercaptoethanol
• 110 µg/ml sodium pyruvate
• 4.5 mg/ml D-glucose
• Is supplemented with 1000 units/ml recombinant
  murine leukemia inhibitory factor (LIF)

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• LIF was withdrawn

• The cells were allowed to aggregate

• The DMEM was changed every other day throughout
  the culture

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Northern Blot Analysis

• Total RNA prepared from undifferentiated ES
  cells, preaggregation ES cells, and EBs of
  various stages

• 1 whole dish of EBs was used for isolation of
  total RNA

• Samples were collected from undifferentiated ES
  cells, preaggregation phase cells, and EBs at
  days 0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, and 18

• RNA electrophoresed and transferred to Hybond-N
  membranes

• Hybridizations performed and signals detected

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RT-PCR

• Total RNA from various stages, and adult mouse
  liver was treated with RNase-free DNase

• cDNA synthesis

• Parallel reaction performed without reverse
  transcriptase to assess presence of DNA
  contamination

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Whole-mount in situ hybridization sectioning of
EBs

• These experiments were carried out as described
  by Sasaki and Hogan

• Stained EBs were refixed in paraformaldehyde and
  glutaraldahyde for 30 minutes

• Then washed twice with PPT and placed in molten
  agarose, solidified, then sectioned

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Molecular Probes

• Xho I fragment of R1 used as ?-fetoprotein (AFP)
  probe

• A fragment of Sma I Sac I fragment of pHF22.1
  used as a hepatocyte nuclear factor (HNF) probe

• A 660-bp Pst I Pvu II fragment of pmPA1 was
  subcloned and used as a transthyretin (TTR) cDNA
  probe

• A variant form of HNF1 (vHNF1) probe was
  synthesized by PCR using adult mouse liver cDNA
  as a template

• Serum albumen (ALB) probe was also made by PCR

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Results

• vHNF1 and HNF4 transcript levels were very low in
  the undifferentiated, preaggregation and day 0
  EBs, then abruptly incresed at day 1

• HNF3? began weak, but the expression level
  increased after day 1

• TTR expression was still low at day 1, but
  increased rapidly thereafter

• These results demonstrate that the activation of
  vHNF1, and HNF4 or HNF3? preceded the rise in TTR
  expression, beginning at day 1

• At day 1, ES cells formed small compact cell
  aggregates, lacking morphologically distinct cell
  populations

• Elevation of TTR levels around day 3 coincide
  with the appearance of primitive endodermal cells

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These data show that all the serum protein genes
were activated at different stages, followed by a
strong increase in expression, and all the
transcription factor genes were not active in the
undifferentiated stem cells, and began to be
expressed in early phase differentiation.
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• The results of the northern blot and RT-PCR
  analyses demonstrated that endodermal cell
  differentiation occurred during the EB development

• Two types of populations were expressed by day 5

• Yolk-sac-like structures
• Hematopoietic cells

• The TTR messages were found only in the
  population of the EBs which began to form
  yolk-sac-like structures and were only found in
  the outer endodermal layer of the yolk-sac-like
  structures

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This suggests that at least one of the endoderm
marker genes is predominantly expressed in the
outer layer of the yolk-sac-like structure during
EB development
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The order of gene expression for the in vivo
studies is similar to that found in EB formation
in vitro
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Conclusion

• These data as a whole strongly suggest that
  development of EBs in vitro closely resembles the
  sequence of in vivo (normal) development of
  visceral yolk sac endoderm.

• The ES cell in vitro differentiation system
  should be useful for analyzing molecular events
  concerning both extraembryonic and embryonic
  endoderm differentiation processes

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The End