Stempeutics Research Pvt. Ltd., Manipal Hospital, Bangalore-560 017, India.
Curr Stem Cell Res Ther. 2010 Mar;5(1):2-12. doi: 10.2174/157488810790442769.
Human embryonic stem cells (hESC) differentiate spontaneously in culture and develop a complex microenvironment comprising of autologously derived niche that in turn supports their pluripotency. The basic hypothesis that we deal with is that hESCs undergoing differentiation, sequentially generate trophectoderm and endoderm lineages and thereafter influence further events through the production of growth factors. These factors control the fate of hESCs either by promoting or retarding the recruitment of new cells in the differentiation program. This scenario therefore represents an analog of the in vivo situation in which extra-embryonic tissues influence the behavior of the inner cell mass (ICM). The premise of the paper is the Rho kinase inhibitor Y27632 that can spatiotemporally alter this balance between pluripotency and differentiation. To evaluate the composition and inclination of lineage specification during spontaneous differentiation, we have studied the hESC colonies and their surrounding niche as interdependent entities. We show that the population of fibroblastic niche that surrounds hESC colonies co-expresses trophectoderm and niche cell markers including SSEA1, hCG, progesterone, HAND1, pSmad1 and FGFR1 as early as day 4. A sudden increase in the expression of GATA4 and AFP secretion indicated putative endoderm formation on day 6 in both control and Y27632 treated cultures. On day 6, 20 microM of Y27632 supplementation significantly reduced the trophectoderm-like niche population without affecting endoderm formation, enhanced the average size and number of hESC colonies, decreased IGF1 secretion thereby improving the pluripotency. Overall our findings support the afore mentioned hypothesis and demonstrate that closely packed epithelial trophectoderm-like cells bordering the hESC colonies present an initial and imminent localized niche which is spatiotemporally regulated. Such advances in understanding the behavior and modulation of hESC and its surrounding niche would facilitate better differentiation protocols for applications in regenerative medicine and drug screening.
人胚胎干细胞（hESC）在培养中自发分化，并形成一个复杂的微环境，其中包括自体衍生的小生境，小生境反过来支持其多能性。我们处理的基本假设是，hESC 在分化过程中，依次产生滋养外胚层和内胚层谱系，然后通过产生生长因子进一步影响后续事件。这些因子通过促进或延缓新细胞在分化程序中的募集来控制 hESC 的命运。因此，这种情况代表了一种类似体内的情况，即胚胎外组织影响内细胞团（ICM）的行为。本文的前提是 Rho 激酶抑制剂 Y27632，它可以时空改变多能性和分化之间的这种平衡。为了评估自发分化过程中谱系特化的组成和倾向，我们将 hESC 集落及其周围小生境作为相互依存的实体进行了研究。我们表明，早在第 4 天，围绕 hESC 集落的成纤维细胞小生境的群体就共同表达滋养外胚层和小生境细胞标记物，包括 SSEA1、hCG、孕酮、HAND1、pSmad1 和 FGFR1。在对照和 Y27632 处理的培养物中，GATA4 的表达突然增加和 AFP 分泌表明潜在的内胚层形成发生在第 6 天。在第 6 天，补充 20μM 的 Y27632 可显著减少滋养外胚层样小生境群体，而不影响内胚层形成，增强 hESC 集落的平均大小和数量，减少 IGF1 分泌，从而提高多能性。总的来说，我们的研究结果支持上述假设，并表明紧密堆积的上皮样滋养外胚层样细胞边界 hESC 集落呈现出初始和即将出现的局部小生境，该小生境受到时空调节。对 hESC 及其周围小生境的行为和调节的深入了解将有助于更好的分化方案，应用于再生医学和药物筛选。
