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  • Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform.

Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform.

Journal of biomedical science (2009-09-24)
Kavitha Sivasubramaiyan, Swapnil Totey, Vijay Bhat, Satish M Totey, Kaushik Deb
ABSTRACT

Trophoblast differentiation and formation of the placenta are important events linked to post-implantation embryonic development. Models mimicking the biology of trophoblast differentiation in a post-implantation maternal microenvironment are needed for understanding disorders like placental-ischemia or for applications in drug-screening, and would help in overcoming the ethical impasse on using human embryos for such research. Here we attempt to create such a model by using embryoid bodies (EBs) and a biomimetic platform composed of a bilayer of fibronectin and gelatin on top of low-melting agarose. Using this model we test the hypothesis that cystic-EBs (day 30) that resemble blastocysts morphologically, are better sources as compared to noncytic EBs (day 10), for functional trophoblast differentiation; and that the Rho kinases inhibitor Y27632 can enhance this differentiation. Non/cytic EBs with/out Y27632 were grown on this platform for 28 days, and screened from secretion and expression of trophoblast and other lineage markers using ECLIA, RT-PCR, and Immunofluorescence. All EBs attached on this surface and rapidly proliferated into hCG and progesterone (P2) secreting functional trophoblast cells. However, the cells derived from cytic-EBs and cytic-EBs+ Y27632 showed the maximum secretion of these hormones and expressed IGF2, supporting our hypothesis. Also Y27632 reduced extraembryonic endoderm and trophoblast lineage differentiation from early noncystic-EBs, whereas, it specifically enhanced the induction of trophoblast and multinucleated syncitiotrophoblast differentiation from late cystic-EBs. In vivo trophoblast differentiation can be replicated in fibronectin based biomaterials, using cytic-EBs and by maneuvering the Rho-ROCK pathways. Response of EBs to a compound may vary temporally, and determination of their right stage is crucial for applications in directed-differentiation or drug-screening.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Paraformaldehyde, powder, 95%
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Paraformaldehyde, reagent grade, crystalline
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Agarose, low gelling temperature, BioReagent, for molecular biology
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Taq DNA Polymerase from Thermus aquaticus, with 10× PCR reaction buffer containing MgCl2
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Agarose, low gelling temperature, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture
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Paraformaldehyde, prilled, 95%
Sigma-Aldrich
Taq DNA Polymerase from Thermus aquaticus, with 10× PCR reaction buffer without MgCl2
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Agarose, low gelling temperature, Type VII-A