Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform.
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.
Sivasubramaiyan
Kavitha
K
Stempeutics Research Pvt Ltd,, Air Port Road, Bangalore KA 560017, India. kavitha.s@mirm.edu
Totey
Swapnil
S
Bhat
Vijay
V
Totey
Satish M
SM
Deb
Kaushik
K
D000577:Amides / Q000494:pharmacology*; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line / Q000187:drug effects; D019070:Cell Lineage; D016764:Cell Polarity / Q000187:drug effects; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology*; D047108:Embryonic Development / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects; D004707:Endoderm / Q000187:drug effects; D005353:Fibronectins; D005780:Gelatin; D006801:Humans; D008954:Models, Biological; D009940:Organoids / Q000166:cytology / Q000187:drug effects*; D011374:Progesterone / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D012685:Sepharose; D014327:Trophoblasts / Q000166:cytology*; D000509:alpha-Fetoproteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
D000577:Amides; D006063:Chorionic Gonadotropin; D003470:Culture Media; D005353:Fibronectins; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D000509:alpha-Fetoproteins; C108830:Y 27632; D011374:Progesterone; D005780:Gelatin; D012685:Sepharose; D054460:rho-Associated Kinases