Paracrine and Epigenetic Control of Trophectoderm Differentiation from Human Embryonic Stem Cells: The Role of Bone Morphogenic Protein 4 and Histone Deacetylases

Erb, Teresa M.; Schneider, Corinne; Mucko, S.E.; Sanfilippo, Joseph S.; Lowry, Nathan; Desai, Mihir A.; Mangoubi, Rami; Leuba, Sanford H.; Sammak, Paul J.

doi:10.1089/scd.2010.0281

Cited by 46 publications

(45 citation statements)

References 90 publications

(134 reference statements)

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“…1B). This phenomenon has been described earlier (1,(9)(10)(11). However, when inhibitors [A83-01 (A) and PD173074 (P)] that block the SMAD2/3 and MEK1/2 signaling pathways, respectively, were added simultaneously with BMP4 (BMP4/A/P), the ESC phenotype of the colonies was lost within 48 h. The transition to a flattened epithelium had progressed completely and quite uniformly throughout all colonies by this time point.…”

Section: Resultssupporting

confidence: 71%

“…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)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(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)(15)(16)(17).…”

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confidence: 99%

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

Amita

Adachi

Alexenko

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

241

307

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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...

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Section: Resultssupporting

confidence: 71%

mentioning

confidence: 99%

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

Amita

Adachi

Alexenko

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

241

307

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“…One previous study developed a minimal medium for trophoblast induction but also used high BMP4 concentrations and focused on the evaluation of CDX2 as the primary TE marker (17). Because high-dose BMP4 also can induce mesoderm (23), we asked whether, in that minimal medium (17), low-dose BMP4 (10 ng/mL) by itself is able to initiate trophoblast differentiation of hPSCs into CTBs, as evaluated by a comprehensive panel of markers, including p63 and CDX2. We initially used WA09/H9 ES cells (ESCs) but also tested this protocol on WA01/H1 and SIVF21 ESCs as well as on induced pluripotent stem cells (iPSCs) derived in the L.C.L.…”

Section: Resultsmentioning

confidence: 99%

“…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…”

mentioning

confidence: 99%

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

Horii

Wakeland

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

136

164

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Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6-8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2 + /p63 + CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2 + /p63 + CTB stem-like cells. These cells can be replated and further differentiated into STB-and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G + EVTs in an hypoxiainducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.human pluripotent stem cells | cytotrophoblast | extravillous trophoblast | syncytiotrophoblast | placenta

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“…For trophectoderm differentiation, hPSCs were first treated with a StemPro-based basal medium (Erb et al, 2011) for 2 days, then cultured in feeder-conditioned medium supplemented with 10 ng/ml human BMP4 (StemRD) for the indicated number of days.…”

Section: Human Pluripotent Stem Cell Culture and Differentiationmentioning

confidence: 99%

BMP4-directed trophoblast differentiation of human embryonic stem cells is mediated through a ΔNp63+ cytotrophoblast stem cell state

et al. 2013

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SUMMARYThe placenta is a transient organ that is necessary for proper fetal development. Its main functional component is the trophoblast, which is derived from extra-embryonic ectoderm. Little is known about early trophoblast differentiation in the human embryo, owing to lack of a proper in vitro model system. Human embryonic stem cells (hESCs) differentiate into functional trophoblast following BMP4 treatment in the presence of feeder-conditioned media; however, this model has not been widely accepted, in part owing to a lack of proof for a trophoblast progenitor population. We have previously shown that p63, a member of the p53 family of nuclear proteins, is expressed in proliferative cytotrophoblast (CTB), precursors to terminally differentiated syncytiotrophoblast (STB) in chorionic villi and extravillous trophoblast (EVT) at the implantation site. Here, we show that BMP4-treated hESCs differentiate into bona fide CTB by direct comparison with primary human placental tissues and isolated CTB through gene expression profiling. We show that, in primary CTB, p63 levels are reduced as cells differentiate into STB, and that forced expression of p63 maintains cyclin B1 and inhibits STB differentiation. We also establish that, similar to in vivo events, hESC differentiation into trophoblast is characterized by a p63 + /KRT7 + CTB stem cell state, followed by formation of functional KLF4 + STB and HLA-G + EVT. Finally, we illustrate that downregulation of p63 by shRNA inhibits differentiation of hESCs into functional trophoblast. Taken together, our results establish that BMP4-treated hESCs are an excellent model of human trophoblast differentiation, closely mimicking the in vivo progression from p63 + CTB stem cells to terminally differentiated trophoblast subtypes.

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Paracrine and Epigenetic Control of Trophectoderm Differentiation from Human Embryonic Stem Cells: The Role of Bone Morphogenic Protein 4 and Histone Deacetylases

Cited by 46 publications

References 90 publications

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

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

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

BMP4-directed trophoblast differentiation of human embryonic stem cells is mediated through a ΔNp63+ cytotrophoblast stem cell state

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