Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform |
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Authors: | Kavitha Sivasubramaiyan Swapnil Totey Vijay Bhat Satish M Totey Kaushik Deb |
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Institution: | (1) Stempeutics Research Pvt Ltd., Air Port Road, 560017 Bangalore, KA, India;(2) Stepeutics Research Malaysia Sdn Bhd, Kuala Lumpur, Malaysia;(3) Department of Biochemistry, Manipal Hospital Diagnostic Services, 560017 Bangalore, KA, India |
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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. |
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