They do not readily survive single-cell dispersion by proteinases rather, they must be passaged by either mechanical breakage of colonies after dispase treatment or gentle dissociation with chelating agents in presence of Rho-associated kinase (ROCK) inhibitors to small clusters of cells. hESCs and iPSCs are maintained with activators of two signaling pathways: the BMP receptor type-1A (BMPR1A) (ALK3) pathway via ACTIVIN and the mitogen-activated protein kinase kinase/ERK signaling pathway via FGF2 ( 6– 8). Human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) form flattened colonies that resemble, and are considered functionally comparable to, pluripotent cells derived from the mouse epiblast, or “primed”-type stem cells ( 1– 5). They may be passaged by dispersal to single cells with trypsin and can be maintained on a gelatin substratum. Mouse ES cells, the “naive” type, are obtained from outgrowths of the inner cell mass/early epiblast of blastocysts ( 1) and are dependent on the growth factor leukemia inhibitory factor (LIF) for maintenance of pluripotency. The results may have implications for regulation of lineage decisions in the early embryo.
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. 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.
In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). 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 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. 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. Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling.
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