1. Embryoid Bodies From Embryonic Stem Cells In-vitro Robert Christensen Biology Department Eastern Connecticut State University

2. 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

3. Introduction Cell lineages arise during development Ectodermal tissues Mesodermal tissues Endodermal tissues EBs resemble the embryo of the egg- cylinder stage Neuronal cells Cardiac muscle cells Hematopoietic cells Yolk sac cells ES cells have full developmental potential EBs from ES cells EBs consist of: Later stages EBs are composed of:

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

5. 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)

6. LIF was withdrawn The cells were allowed to aggregate The DMEM was changed every other day throughout the culture

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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.

13. 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

14. 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

15. The order of gene expression for the in vivo studies is similar to that found in EB formation in vitro

16. 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

17. The End