Download Embryoid Bodies From Embryonic Stem Cells In

Embryoid Bodies From
Embryonic Stem Cells In-vitro
Robert Christensen
Biology Department
Eastern Connecticut State University
Endoderm–Specific Gene Expression in
Embryonic Stem–Cells 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
Introduction
•
•
•
•
Cell lineages arise during development
ES cells have full developmental potential
EBs from ES cells
EBs consist of:
•Ectodermal tissues
•Mesodermal tissues
•Endodermal tissues
• EBs resemble the embryo of the egg- cylinder stage
• Later stages EBs are composed of:
•Neuronal cells
•Cardiac muscle cells
•Hematopoietic cells
•Yolk sac cells
•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.
Materials and Methods
Cell cultures
•ES cell line, D3, cultured
•Incubated 3 days in Dulbecco’s 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)
•LIF was withdrawn
•The cells were allowed to aggregate
•The DMEM was changed every other day throughout the culture
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
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
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
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
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
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.
•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
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
The order of gene expression for the in vivo studies is similar to
that found in EB formation in vitro
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
The End