Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4

Abstract

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development, but the soundness of this model has been challenged by others, who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7+ within 48 h, with minimal expression of mesoderm markers, including T (Brachyury). Instead, they begin to express a series of trophoblast markers, including HLA-G, demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium, and, over time, produce extensive amounts of human chorionic gonadotropin, progesterone, placental growth factor, and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling, which at day 2 provide colonies that are entirely KRT7 + and in which the majority of cells are transiently CDX2 + . Colonies grown on two chemically defined media, including the one in which BMP4 was reported to drive mesoderm formation, also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts.A popular model for examining the early commitment of cells to the trophoblast (TR) lineage is based on the initial observation of Xu et al. (1), who noted that a group of related factors in the TGF-β family, especially bone morphogenic protein 4 (BMP4), was capable of causing human ES cells (hESC) to differentiate efficiently to TRs. This differentiation occurred without extensive generation of mesoderm, endoderm, and ectoderm derivatives, as judged by microarray analysis of transcribed genes, although a low level of expression of genes characteristic of mesoderm and endoderm did occur. This model has become widely used (2-13) to study an aspect of early human development that is not easily addressed otherwise because of lack of access to human embryos. Over the course of these studies it was demonstrated that the key to obtaining differentiation primarily to TR rather than to other lineages when using BMP4 as the triggering agent was to exclude FGF2, a factor required for maintenance of hESC (14-17). When BMP4 is provided simultaneously with FGF2, the morphological transition of the cells is altered (10), and the colonies begin to form a range of mesoderm and endoderm derivatives in addition to TR (18). This effect is probably achieved by FGF2 signaling through the MEK/ERK pathway, thereby preserving NANOG expression (19,20). This body of work suggests that optimal differentiation to TR can be achieved best by maximizing BMP4 signaling while simultaneous...

Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease

“…In contrast, ELF5 is not expressed in the TE of the human preimplantation blastocyst (13). As in previous studies (20), we do see induction of ELF5 following BMP4 treatment; however, this induction is weak (1.5-to twofold in all lines tested) and does not reach the levels of expression seen in postimplantation CTBs (24). Therefore, it is possible that the hPSC-derived CTBs are more similar to preimplantation TE than to early postimplantation CTBs.…”

“…Nevertheless, follow-up studies using more defined culture conditions have confirmed the identity of these cells as trophoblasts (20).…”

“…Since 2002, when Xu et al (15) first published the finding that bone morphogenetic protein 4 (BMP4) induces the expression of trophoblast-related genes in hPSCs, multiple groups have used these cells as a model for studying trophoblast lineage specification (16)(17)(18)(19)(20)(21)(22). The majority of these studies, including our own (21), have used BMP4…”

Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas

“…used in combination with two small compounds, the activin A signaling inhibitor A83-01, and the FGF2 signaling inhibitor PD173074 (BMP4/A83-01/PD173074; BAP treatment), the process becomes more efficient and synchronous, such that by 48 h, almost all of the cells in the colonies have become transiently positive for the transcription factor CDX2 and completely positive for the trophoblast marker KRT7 (14,17). By day 6 of treatment, areas of syncytium emerge, and the culture medium begins to accumulate significant quantities of the placental hormones, chorionic gonadotropin (CG), placental growth factor (PGF), and progesterone (14).…”

“…A third approach has been to purify cytotrophoblast populations from whole placentas, usually from term collections, which, when cultured appropriately, fuse and form a multinucleated syncytium exhibiting many of the physiological features expected of STB within 2-3 d (12). An additional, fourth approach for generating what appears to be STB, is from human pluripotent stem cells, which, in absence of fibroblast growth factor-2 (FGF2) and in a process primed by the presence of the growth factor bone morphogenetic protein-4 (BMP-4), form areas of syncytium within the colonies (13)(14)(15). An up-regulation of genes expressed in placental syncytium accompanies this process of progressive differentiation (13,16).…”