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Bone Morphogenetic Protein-4 Affects Both Trophoblast and Non-Trophoblast Lineage-Associated Gene Expression in Human Embryonic Stem Cells

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Bone Morphogenetic Protein-4 Affects Both Trophoblast and Non-Trophoblast Lineage-Associated Gene Expression in Human Embryonic Stem Cells Vol.2, No.4, 163-175 (2012) Stem Cell Discovery http://dx.doi.org/10.4236/scd.2012.24021 Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells Margaret L. Shirley1,2*, Alison Venable1*, Raj R. Rao3, Nolan L. Boyd4, Steven L. Stice1,5,6, David Puett1#, Prema Narayan7# 1Department of Biochemistry and Molecular Biology, University of Georgia, Athens, USA; #Corresponding Author: [email protected] 2Department of Psychiatry, University of California, San Francisco, USA 3Department of Chemical and Life Science Engineering, School of Engineering, Virginia Commonwealth University, Richmond, USA 4Cardiovascular Innovation Institute, University of Louisville, Louisville, USA 5Regenerative Bioscience Center, University of Georgia, Athens, USA 6Department of Animal and Dairy Sciences, University of Georgia, Athens, USA 7Department of Physiology, Southern Illinois University School of Medicine, Carbondale, USA; #Corresponding Author: [email protected] Received 5 May 2012; revised 4 June 2012; accepted 1 July 2012 ABSTRACT cells were obtained. Gene expression by EB was characterized by an up-regulation of a num- Human embryonic stem cells (hESC) can be in- ber of genes associated with trophoblast, ecto- duced to differentiate to trophoblast by bone derm, endoderm, and mesoderm, and the pro- morphogenetic proteins (BMPs) and by aggre- duction of hCG and progesterone confirmed that gation to form embryoid bodies (EB), but there trophoblast-like cells were formed. These re- are many differences and controversies regard- sults suggest that, in the presence of FGF-2, ing the nature of the differentiated cells. Our BG02 cells respond to BMP4 to yield tropho- goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of blast-like cells, which are also obtained upon EB BMP4-plus-basic fibroblast growth factor (FGF-2) formation. Thus, BMP4-mediated differentiation and (b) upon EB formation, and (c) whether the of hESC represents a viable cell system for BMP4 antagonist noggin elicits direct effects on studying early developmental events post-im- gene expression and hormone production in the plantation; however, up-regulation of non-tro- cells. Transcriptome profiling of hESC incubated phoblast genes suggests a somewhat diverse with BMP4/FGF-2 showed a down-regulation of response to BMP4/FGF-2. Noggin altered the pluripotency-associated genes, an up-regulation transcription of a limited number of genes but, of trophoblast-associated genes, and either a not surprisingly, did not lead to secretion of down-regulation or no change in gene expres- hormones. sion for many markers of the three embryonic germ layers. Yet, there was up-regulation of Keywords: Human Embryonic Stem Cells; several genes associated with mesoderm, ec- Trophoblasts; Bone Morphogenetic Protein-4; toderm, and endoderm, strongly suggesting that Embryoid Bodies; Noggin differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous 1. INTRODUCTION cell type. Several genes, heretofore unreported, were identified that are altered in hESC in re- In the human blastocyst, the first step of differentiation sponse to BMP4-mediated differentiation. The from the morula, composed of totipotent cells, yields the production of human chorionic gonadotropin inner cell mass and trophoblast. The former differentiates (hCG), progesterone, and estradiol in the dif- into the hypoblast, leading to the extraembryonic endo- ferentiated cells confirmed that trophoblast-like derm and the epiblast, that differentiates to give the am- niotic ectoderm and the primitive ectoderm. The primi- * These authors contributed equally to this work. tive ectoderm, in turn, differentiates into the embryonic Copyright © 2012 SciRes. OPEN ACCESS 164 M. L. Shirley et al. / Stem Cell Discovery 2 (2012) 163-175 ectoderm and the primitive streak, the latter giving rise to gene markers have been noted in BMP-treated hESCs extraembryonic mesoderm, primitive mesoderm, and em- [18,21]. Explanations for many of these controversial bryonic endoderm. The trophectoderm is composed of reports were recently provided by two groups. Yu et al. epithelial cells and differentiates into several lineages. [22] reported that bFGF (FGF-2), which acts via the The trophoblast precursor cells begin rapid proliferation MEK-ERK pathway, redirects BMP4-mediated differen- into cytotrophoblasts, with some fusing to form multinu- tiation from trophectoderm to mesendoderm as judged by cleate syncytiotrophoblasts, a terminally differentiated the expression of brachyury. The activated MEK-ERK trophoblast cell; in addition, villous and extravillous cy- pathway sustains NANOG expression leading to a totrophoblasts are formed [1-3]. One of the hallmarks of bFGF-independent induction of mesendoderm by BMP4. trophoblast differentiation is the production of the hete- Another recent paper concluded that in the cooperative rodimeric glycoprotein hormone, human chorionic gona- presence of FGF2, BMP4 (that acts through brachyury dotropin (hCG), that is secreted throughout gestation, and CDX2) leads to the induction of mesoderm, not tro- being essential for progesterone production by the corpus phoblast [39]. luteum in the first trimester of human pregnancy [4,5]. The present study was undertaken with the goals of: 1) There is a paucity of adequate cell models for studying extending the previous reports that were based on a va- early trophoblast development, and a reliable system will riety of cell lines, different culture conditions, and at greatly facilitate progress in the area of human reproduc- times conflicting results, and 2) to critically examine the tion. trophoblast and non-trophoblast BMP-regulated genes, Recent studies have shown that human embryonic particularly since a number of the non-trophoblast BMP- stem cells (hESCs) can undergo differentiation into tro- regulated genes have been noted in the earlier studies. phoblast-like cells spontaneously from colonies [6], We chose to use the hESC line, BG02 (karyotype 46, growth of the cells beyond confluence [7], or embryoid XY), that has not been thoroughly studied in hESC dif- bodies (EBs) [3,8-12]. Similarly, trophoblast-like cells ferentiation to trophoblast. This cell line was established can be obtained from hESCs via induced differentiation from a 6-day embryo with an embryo grade of 3CC [7]. from the bone morphogenetic proteins (BMPs) 2, 4, and Herein, BMP4, in the presence of FGF2, was incubated 7 [13-24]. Fortunately, timely reviews are available [25- with adherent colonies of BG02 cells to initiate tro- 28]. Others have reported that RNAi-mediated knock- phoblast differentiation. Identical studies were also done down of Oct4 in hESCs can also lead to trophoblast-like in the presence of the BMP4 antagonist noggin, since it cells [29,30]. The BMPs, members of the transforming has been shown that noggin alters hESC gene expression growth factor-b (TGFb) superfamily, function to regulate and morphology, perhaps leading to differentiation to- many aspects of development which act by binding to ward early neuroectoderm [18]. In addition, trophoblast cell surface serine/threonine kinase receptors that, in turn, differentiation was initiated by formation of EBs. Parti- phosphorylate particular Smads, thus enabling them to cular emphasis was placed on quantitative profiling of a enter the nucleus and act as transcriptional regulators variety of genes via qRT-PCR, and measurements were [31-34]. made to determine hCG and steroid hormone production These cellular models for trophectoderm differentia- by the cells and EBs. Our results have many similarities tion begin to fill a long-awaited need for new systems to with the findings of others, including for example the study early development, particularly in view of the ma- down-regulation of pluripotency genes, the up-regulation jor differences between human and mouse trophoblast of trophoblast genes, and placental hormone production, [35-37]. Yet, the characteristics of the human trophoblast but there are also notable differences, particularly with cells obtained by BMP-mediated differentiation vary, the up-regulation of certain genes associated with the three sometimes quite significantly, depending upon the cell primary germ layers. type, culture conditions (including the presence or ab- sence of growth factors, particularly FGF2), and mode of 2. MATERIALS AND METHODS differentiation [10,18,38]. Moreover, in a number of in- stances there are considerable differences and controver- 2.1. Maintenance of Undifferentiated Cells sies regarding the product(s) of differentiation obtained In order to ensure pluripotency, the BG02 cells, ob- with BMP. For example, BMP4-mediated differentiation tained with proper authorization from Bresagen, Athens, was found to yield little evidence of trophoblast-like GA, were manually passaged every 2 - 3 days onto mito- cells under standard culture conditions [38]. Another stu- tically-inactivated mouse embryonic feeder (MEF) layers dy found that 14 genes were highly up-regulated as de- derived from E13.5 mouse fetuses. The MEF layer was termined by microarrays, in addition to many others [21], removed from the hESC colony, which was then gently while another report did not identify six of these particu- dispersed with the colony pieces being transferred to lar genes [17]. Also, differences in cell morphology and another 10 cm MEF-containing plate and treated with Copyright
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