Yang Ying, Adachi Katsuyuki, Sheridan Megan A, Alexenko Andrei P, Schust Danny J, Schulz Laura C, Ezashi Toshihiko, Roberts R Michael
Division of Animal Sciences, Bond Life Sciences Center and.
Department of Obstetrics, Gynecology, and Women's Health, University of Missouri, Columbia, MO 65212.
Proc Natl Acad Sci U S A. 2015 May 5;112(18):E2337-46. doi: 10.1073/pnas.1504778112. Epub 2015 Apr 13.
Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.
人类多能干细胞（PSC）表现出上胚层型多能性，这种多能性通过激活素/成纤维细胞生长因子2（ACTIVIN/FGF2）信号得以维持。在此，我们报告人类胚胎干细胞（hESC）和诱导多能干细胞（iPSC）在短暂（24 - 36小时）暴露于骨形态发生蛋白4（BMP4）以及激活素信号抑制剂（A83 - 01）和成纤维细胞生长因子2抑制剂（PD173074）后获得了一种独特的干细胞表型。随后通过胰蛋白酶消化并回收能够在低浓度而非高浓度FGF2的标准人类PSC培养基中的明胶基质上生长的集落。这些自我更新的细胞系对CDX2染色弱阳性，对NANOG染色强阳性，可在基质胶或明胶上进行克隆扩增，并且在两种基质上其形态均与人类PSC祖细胞不同，但仍符合多能性的标准体外标准。它们在免疫缺陷小鼠中形成分化良好的畸胎瘤，这些畸胎瘤会向宿主小鼠分泌人绒毛膜促性腺激素（hCG），并且包含小面积的滋养层样细胞。这些细胞具有与它们所来源的人类PSC不同的转录组谱（包括NANOG、LEFTY1和LEFTY2的更高表达）。在缺乏FGF2的非条件培养基中，集落会自发地沿着多个谱系分化，包括滋养层。在缺乏FGF2和BMP4的情况下，它们对PD173074的反应是转化为滋养层，尤其是合体滋养层，而A83 - 01/PD173074组合则有利于增加人绒毛膜促性腺激素（HLA - G）的表达，HLA - G是绒毛外滋养层的标志物。总之，这些数据表明这些细胞系表现出全能潜力，并且BMP4可以将人类PSC诱导至一种允许滋养层发育的自我更新替代状态。这些结果可能对早期胚胎中谱系决定的调控具有重要意义。
