Abstract
AbstractDivision of the human zygote leads to the formation of the blastocyst that contains human embryonic stem cells (hESC) which develop into the embryo. Little is known about the physiological signals that direct hESC division and differentiation during early embryogenesis. A number of growth factors, including the pregnancy-associated hormone human chorionic gonadotropin (hCG), are secreted by trophoblasts^1-3^ that lie adjacent to the embryoblast in the blastocyst, but it is not known whether these growth factors directly signal the epiblast. Here we show that hCG promotes the division of embryoblast-derived inner mass cells (hESC), and their differentiation during blastulation and neurulation. Inhibition of LH/hCG receptor (LHCGR) signaling with P-antisense oligonucleotides suppresses hESC proliferation. Similarly, hESC proliferation can be blocked using an antibody against the extracellular activation site of LHCGR, an effect that is reversed by treatment with hCG. hCG treatment rapidly upregulates steroidogenic acute regulatory protein-mediated cholesterol transport and the synthesis of progesterone, a neurogenic steroid^4,5^. Treatment of hESC colonies with progesterone induces neurulation as demonstrated by the expression of nestin and the formation of columnar neuroectodermal cells that organize into neural tube-like rosettes. Suppression of progesterone signaling by withdrawing progesterone or treating with the progesterone receptor antagonist RU-486 inhibits the differentiation of hESC colonies into embryoid bodies (blastulation) and rosettes (neurulation). These results explain the default pathway of hESC differentiation towards a neural stem cell fate in vitro. Collectively, our findings implicate trophoblastic hCG secretion and signaling via LHCGR on the adjacent embryoblast in the induction of hESC proliferation and differentiation into blastocysts and neurula. This paracrine/juxtacrine signaling by extraembryonic tissues is the commencement of trophic support by placental tissues in the growth and development of the human embryo.
Highlights
Given the close spatial localization of the developing trophoblast layer to the epiblast, it is conceivable that trophoblast-associated hormones directly signal the growth and development of the epiblast
To examine the functionality of trophoblastic signaling to the epiblast, we examined if the LH/hCG receptor (LHCGR) was expressed by pluripotent human embryonic stem cells (hESC)
Treatment of hESC with a physiologically relevant concentration of human chorionic gonadotropin (hCG) (500 mIU/mL) in growth factor-free TESR1 culture media resulted in a 3.3-fold increase in cell proliferation after 6 d (Fig. 1b), a response that did not vary with hCG concentration (5-50,000 mIU/mL; data not shown)
Summary
HCG treatment induced nestin expression (205-kDa variant) in hESC (Fig. 4e), indicating endogenous gonadotropin production by hESC (Fig. 1) or trophoblastic cells[2] may be sufficient for NPC formation, thereby explaining the extrinsic hormonal signals regulating the ‘default pathway’ of hESC differentiation into neuronal lineages[25] These results suggest that trophoblastic hCG production adjacent to the embryoblast is required for trophoblast steroidogenesis and attachment to the uterine wall, and for signaling normal growth and development of the epiblast. There exists a critical molecular signaling link between trophoblastic (and/or maternal) hormone production and early embryonic growth and development
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