Andreia S. Bernardo

BRACHYURY and CDX2 Mediate BMP-Induced Differentiation of Human and Mouse Pluripotent Stem Cells into Embryonic and Extraembryonic Lineages

Summary BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.

Generation of human vascular smooth muscle subtypes provides insight into embryological origin–dependent disease susceptibility

Heterogeneity of embryological origins is a hallmark of vascular smooth muscle cells (SMCs) and may influence the development of vascular disease. Differentiation of human pluripotent stem cells (hPSCs) into developmental origin–specific SMC subtypes remains elusive. Here we describe a chemically defined protocol in which hPSCs were initially induced to form neuroectoderm, lateral plate mesoderm or paraxial mesoderm. These intermediate populations were further differentiated toward SMCs (>80% MYH11+ and ACTA2+), which displayed contractile ability in response to vasoconstrictors and invested perivascular regions in vivo. Derived SMC subtypes recapitulated the unique proliferative and secretory responses to cytokines previously documented in studies using aortic SMCs of distinct origins. Notably, this system predicted increased extracellular matrix degradation by SMCs derived from lateral plate mesoderm, which was confirmed using rat aortic SMCs from corresponding origins. This differentiation approach will have broad applications in modeling origin-dependent disease susceptibility and in developing bioengineered vascular grafts for regenerative medicine.

Pancreatic transcription factors and their role in the birth, life and survival of the pancreatic β cell

In recent years major progress has been made in understanding the role of transcription factors in the development of the endocrine pancreas in the mouse. Here we describe how a number of these transcription factors play a role in maintaining the differentiated phenotype of the beta cell, and in the mechanisms that allow the beta cell to adapt to changing metabolic demands that occur throughout life. Amongst these factors, Pdx1 plays a critical role in defining the region of the primitive gut that will form the pancreas, Ngn3 expression drives cells towards an endocrine lineage, and a number of additional proteins including Pdx1, in a second wave of expression, Pax4, NeuroD1/beta2, and MafA act as beta cell differentiation factors. In the mature beta cell Pdx1, MafA, beta2, and Nkx2.2 play important roles in regulating expression of insulin and to some extent other genes responsible for maintaining beta cell function. We emphasise here that data from gene expression studies in rodents seldom map on to the known structure of the corresponding human promoters. In the adult the beta cell is particularly susceptible to autoimmune-mediated attack and to the toxic metabolic milieu associated with over-eating, and utilises a number of these transcription factors in its defence. Pdx1 has anti-apoptotic and proliferative activities that help facilitate the maintenance of beta cell mass, while Ngn3 may be involved in the recruitment of progenitor cells, and Pax4 (and possibly HNF1alpha and Hnf4alpha) in the proliferation of beta cells in the adult pancreas. Other transcription factors with a more widespread pattern of expression that play a role in beta cell survival or proliferation include Foxo1, CREB family members, NFAT, FoxM1, Snail and Asc-2.

Biphasic Induction of Pdx1 in Mouse and Human Embryonic Stem Cells Can Mimic Development of Pancreatic β‐Cells

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes. Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic β‐cell development. Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse and human ES cell. By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage‐dependent requirement for this transcription factors activity. Mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas‐like cell phenotype, in which 30% of the cells were β‐cell‐like. Cell markers consistent with the different β‐cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development. Furthermore, in mouse ES‐derived cultures the differentiated β‐like cells secreted C‐peptide (insulin) in response to KCl and 3‐isobutyl‐1‐methylxanthine, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells. Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non‐β‐cells in the developing endocrine pancreas. Collectively, we show that this method of in vitro differentiation provides a template for inducing and studying ES cell differentiation into insulin‐secreting cells. STEM CELLS 2009;27:341–351

Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments. Summary: In differentiating hESCs, BRACHYURY has cell type-specific targets and functions: it acts with SMAD1 to promote mesoderm specification, while in the endoderm it interacts with SMAD2/3.

Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

Directed differentiation of embryonic origin–specific vascular smooth muscle subtypes from human pluripotent stem cells

Vascular smooth muscle cells (SMCs) arise from diverse developmental origins. Regional distribution of vascular diseases may, in part, be attributed to this inherent heterogeneity in SMC lineage. Therefore, systems for generating human SMC subtypes of distinct embryonic origins would represent useful platforms for studying the influence of SMC lineage on the spatial specificity of vascular disease. Here we describe how human pluripotent stem cells can be differentiated into distinct populations of SMC subtypes under chemically defined conditions. The initial stage (days 0–5 or 0–7) begins with the induction of three intermediate lineages: neuroectoderm, lateral plate mesoderm and paraxial mesoderm. Subsequently, these precursor lineages are differentiated into contractile SMCs (days 5–19+). At key stages, the emergence of lineage-specific markers confirms recapitulation of embryonic developmental pathways and generation of functionally distinct SMC subtypes. The ability to derive an unlimited supply of human SMCs will accelerate applications in regenerative medicine and disease modeling.

Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development

Summary Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin/EGFP mouse

In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.5 (E11.5). EGFP expression appeared at E12.5 and increased in intensity through E14.5, E18.5 and post-natal day 1. Flow cytometry was used to quantify and purify the EGFP positive population in the E15.5 pancreas. The purified (96%) EGFP-expressing cells, which represent 20% of the total cell population, were shown by RT/PCR to express exocrine cell markers (amylase and P48) and endocrine cell markers (insulin 1, insulin 2, and Ngn3). They also expressed, at a lower level, PDX-1, Isl-1, and the islet hormones pancreatic polypeptide, glucagon and somatostatin as well as GLUT2, the stem cell marker ABCG2 and PECAM, a marker of endothelial cells. It was further shown by immunocytochemistry of the E15.5 pancreas that EGFP colocalised in separate subpopulations of cells that expressed nestin, insulin and amylase. These results support the conclusion that nestin expressing cells can give rise to both endocrine and exocrine cells. The ability to purify these putative progenitor cells may provide further insights into their properties and function.

Stable methylation at promoters distinguishes epiblast stem cells from embryonic stem cells and the in vivo epiblasts.

Embryonic Stem Cells (ESCs) and Epiblast Stem Cells (EpiSCs) are the in vitro representatives of naïve and primed pluripotency, respectively. It is currently unclear how their epigenomes underpin the phenotypic and molecular characteristics of these distinct pluripotent states. Here, we performed a genome-wide comparison of DNA methylation between ESCs and EpiSCs by MethylCap-Seq. We observe that promoters are preferential targets for methylation in EpiSC compared to ESCs, in particular high CpG island promoters. This is in line with upregulation of the de novo methyltransferases Dnmt3a1 and Dnmt3b in EpiSC, and downregulation of the demethylases Tet1 and Tet2. Remarkably, the observed DNA methylation signature is specific to EpiSCs and differs from that of their in vivo counterpart, the postimplantation epiblast. Using a subset of promoters that are differentially methylated, we show that DNA methylation is established within a few days during in vitro outgrowth of the epiblast, and also occurs when ESCs are converted to EpiSCs in vitro. Once established, this methylation is stable, as ES-like cells obtained by in vitro reversion of EpiSCs display an epigenetic memory that only extensive passaging and sub-cloning are able to almost completely erase.