An efficient method of inducing differentiation of mouse embryonic stem cells into primitive endodermal cells

BackgroundInterleukin-6 (IL6) was recently identified as an embryotrophic factor in bovine embryos, where it acts primarily to mediate inner cell mass (ICM) size. This work explored whether IL6 affects epiblast (EPI) and primitive endoderm (PE) development, the two embryonic lineages generated from the ICM after its formation. Nuclear markers for EPI (NANOG) and PE (GATA6) were used to differentiate the two cell types.ResultsIncreases (P < 0.05) in total ICM cell numbers and PE cell numbers were detected in bovine blastocysts at day 8 and 9 post-fertilization after exposure to 100 ng/ml recombinant bovine IL6. Also, IL6 increased (P < 0.05) the number of undifferentiated ICM cells (cells containing both PE and EPI markers). The effects of IL6 on EPI cell numbers were inconsistent. Studies were also completed to explore the importance of Janus kinase 2 (JAK2)-dependent signaling in bovine PE cells. Definitive activation of STAT3, a downstream target for JAK2, was observed in PE cells. Also, pharmacological inhibition of JAK2 decreased (P < 0.05) PE cell numbers.ConclusionsTo conclude, IL6 manipulates ICM development after EPI/PE cell fates are established. The PE cells are the target for IL6, where a JAK-dependent signal is used to regulate PE numbers.

Cell fate specification begins in preimplantation embryo with the emergence of trophectoderm (TE). Primitive endoderm (PE) is the second extraembryonic lineage to form from cells within the inner cell mass (ICM) during embryogenesis. Ruminant conceptuses elongate dramatically prior to uterine attachment and extensive migration and proliferation of TE and PE is crucial for normal conceptus development and elongation. Work described here provides evidence that FGF2 and potentially other FGFs impact PE development during bovine embryonic development. Bovine blastocyst outgrowths were generated from in vitro-produced embryos using a feeder-layer free culture system containing a Matrigel-coated surface. Individually cultured bovine blastocysts were supplemented with FGF2 at the beginning of culture (day 8 post-IVF) and identification of PE-like cells was evaluated microscopically on days 13 and 15. In controls, only a single blastocyst outgrowth contained what appeared morphologically as PE cells (1.3% of all outgrowths). By contrast, 10.5±6.7%, 19.7±0.28% and 22.4±0.26% of outgrowths contained PE colonies on day 15 when embryos were exposed to 0.5, 5 and 50ng/ml FGF2, respectively. A subset of outgrowths (n=6 TE only and 6 PE-containing) were selected and cultured to day 20, then lineage-specific markers were evaluated using qRT-PCR. Transcripts for IFNT and CDX2 (TE markers) were present in TE outgrowths. Trace amounts of IFNT and CDX2 mRNA were detected in PE colonies (P<0.05) likely because residual TE cells still existed in some of these outgrowths. PE-containing outgrowths contained ample quantities of GATA4 and GATA6 mRNA (putative PE markers) whereas TE outgrowths contained no GATA4 and low amounts of GATA6 mRNA. None of the outgrowths contained NANOG mRNA whereas all TE and PE outgrowths contained OCT4 mRNA. Western blot analysis determined PE-containing outgrowths produced transferrin, a PE product. Moreover, immunoreactive GATA4 localized to nuclei of PE cells but was not detected in TE cells. By contrast, CDX2 localized to TE nuclei but was not detected in PE cells. Continued cultivation and selection for TE and PE yielded pure cell populations (n=6 of each). Differences in relative abundance of FGF receptors (FGFRs) and selective alternatively spliced receptor isoforms were evident between the cell types. Specifically, transcripts for FGFR2b and FGFR3 predominated in TE cells while FGFR1b represented the primary transcript in PE cells. TE and PE cells responded differently to FGF2 treatment. FGF2 supplementation did not affect the mitotic index of TE cells whereas the mitotic index of PE cells increased (P<0.05) following exposure to 5 or 50ng/ml FGF2 (35.1±0.09% and 34.7±0.11%, respectively versus 19.9±0.06% for controls). These observations indicate that the non-TE cells formed with blastocyst exposure to FGF2 are PE, and these cells respond differently to FGF2 than TE. Collectively, this work provides new insight into another vital activity for FGF2 and potentially other FGFs during early embryonic development in bovine. This project was supported by NRICGP number 2008-35203-19106 from the USDA-CSREES. (platform)

Histone deacetylases (HDACs) have a central role in the regulation of gene expression. Here we investigated whether HDAC7 has an impact on embryonic stem (ES) cell differentiation into smooth muscle cells (SMCs). ES cells were seeded on collagen-IV-coated flasks and cultured in the absence of leukemia inhibitory factor in differentiation medium to induce SMC differentiation. Western blots and double-immunofluorescence staining demonstrated that HDAC7 has a parallel expression pattern with SMC marker genes. In ex vivo culture of embryonic cells from SM22-LacZ transgenic mice, overexpression of HDAC7 significantly increased beta-galactosidase-positive cell numbers and enzyme activity, indicating its crucial role in SMC differentiation during embryonic development. We found that HDAC7 undergoes alternative splicing during ES cell differentiation. Platelet-derived growth factor enhanced ES cell differentiation into SMCs through upregulation of HDAC7 splicing. Further experiments revealed that HDAC7 splicing induced SMC differentiation through modulation of the SRF-myocardin complex. These findings suggest that HDAC7 splicing is important for SMC differentiation and vessel formation in embryonic development.

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Transcription factor Krüppel-like factor 5 (Klf5) plays important roles in the formation of the inner cell mass (ICM) and the trophectoderm during embryogenesis, as well as the self-renewal and the differentiation of mouse embryonic stem cells (ESCs). Acetylation of KLF5 has been shown to reverse the transcriptional activity of KLF5 in human epidermal cells and prostate cancer cells. Whether Klf5 acetylation contributes to the lineage specification in the blastocyst and pluripotency maintenance in ESCs remains unexplored. Here, we showed the ubiquitous expression of acetylated Klf5 in the ICM and the trophectoderm, ruling out the possibility that differential acetylation status of Klf5 leads to the lineage specification in the blastocyst. We found that K358Q mutation, mimicking acetylation, enhances the transcriptional activity of Klf5 for pluripotency genes in ESCs, and that K358Q Klf5 is more potent in pluripotency maintenance and in somatic cell reprogramming, compared to K358R Klf5. In ESCs, Klf5 acetylation, stimulated by TGF-β signaling, is involved in enhancing Sox2 expression. Moreover, upon ESC differentiation, acetylation of Klf5 facilitates the suppression of many differentiation genes, except for that K358Q Klf5 activates Cdx2, promoting trophectodermal differentiation. In summary, our results revealed the regulatory functions of Klf5 acetylation in ESC self-renewal and differentiation.

Specification of the epiblast (EPI) and primitive endoderm (PE) in the mouse embryo involves fibroblast growth factor (FGF) signaling through the RAS/MAP kinase pathway. FGFR1 and FGFR2 are thought to mediate this signaling in the inner cell mass (ICM) of the mouse blastocyst and BMP signaling can also influence PE specification. In this study, we further explored the dynamics of FGFR2 expression through an enhanced green fluorescent protein (eGFP) reporter mouse line (FGFR2-eGFP). We observed that FGFR2-eGFP is present in the late 8-cell stage; however, it is absent or reduced in the ICM of early blastocysts. We then statistically correlated eGFP expression with PE and EPI markers GATA6 and NANOG, respectively. We detected that eGFP is weakly correlated with GATA6 in early blastocysts, but this correlation quickly increases as the blastocyst develops. The correlation between eGFP and NANOG decreases throughout blastocyst development. Treatment with FGF from the morula stage onwards did not affect FGFR2-eGFP presence in the ICM of early blastocysts; however, late blastocysts presented FGFR2-eGFP in all cells of the ICM. BMP treatment positively influenced FGFR2-eGFP expression and reduced the number of NANOG-positive cells in late blastocysts. In conclusion, FGFR2 is not strongly associated with PE precursors in the early blastocyst, but it is highly correlated with PE cells as blastocyst development progresses, consistent with the proposed role for FGFR2 in maintenance rather than initiating the PE lineage.

The ability to direct differentiation of mouse embryonic stem (ES) cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA), the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4) both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA) inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR) analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad)-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway.

Embryonic stem (ES) cells may represent an alternative source of functionally intact cardiomyocytes for the causal treatment of cardiovascular diseases. However, this requires cardiac-specific differentiation of stem cells and the selection of pure lineages consisting of early embryonic cardiomyocytes. Therefore, an understanding of the basic mechanisms of heart development is essential for selective differentiation of embryonic stem cells into cardiac cells. The development of cardiac cells from embryonic stem cells is regulated by several soluble factors and signalling molecules together with cardiac specific transcription factors such as the zinc-finger GATA proteins and Nkx-2.5. GATA-4 and Nkx-2.5 seem to be essential for heart development. The use of enhanced green fluorescent protein (EGFP) under the control of cardiac-specific promoters in combination with the ES cell system has allowed for the functional characterisation of cardiac precursor cells. Embryonic stem cell-derived cardiomyocytes developmentally express similar cardiac-specific proteins, ion channels and signalling molecules to that of adult cardiomyocytes. Furthermore, identification of growth factors and signalling molecules under cell culture conditions is crucial for the selective cardiac differentiation of embryonic stem cells. Therefore, serum-free culture conditions have to be established in order to examine the influence of different growth factors and signalling molecules on cardiac development and/or formation from ES cells. Although significant progress has been made in generating cardiac cell lineage by the combination of genetically manipulative methods with selective culture conditions for cell transplantation therapy, one of the remaining future challenges for transplantation in humans is the immunological rejection of the engrafted cardiomyocytes.

Smooth muscle cells (SMCs) are important cell type for regenerative medicine. Previous studies showed that retinoic acid (RA) induces differentiation of SMCs from monolayer-cultured embryonic stem cells (ESCs) with high efficiency. However, the underlying mechanisms are still poorly defined. Here, we identified Wnt signaling as a primary regulator for RA-induced ESC differentiation. The activation of Wnt signaling inhibited the epithelial-mesenchymal transition during ESC differentiation, leading to inhibition of RA-induced SMC differentiation and promoting differentiation of ESCs toward primitive endoderm (PrE) lineage instead, while the inhibition of Wnt signaling promoted RA-induced SMC differentiation. Loss-of-function studies revealed that 7-like 2 (Tcf7l2) was the key transcription factor that Wnt operate through during RA-induced differentiation. Thus, this study revealed that the Tcf7l2-mediated Wnt signaling is a switch in determining the mesoderm/PrE fates in RA-induced ESC differentiation.

Differentiation of embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) govern gene expression patterns by being recruited to target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for acetylation and DNA binding of GATA-4 and its full transcriptional activity as well as for promotion of a transcriptionally active chromatin configuration. However, the roles of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of ES cells into cardiac myocytes remain unknown. In an ES cell model of developing embryoid bodies, an acetylated form of GATA-4 and its DNA binding increased concomitantly with the expression of p300 during the differentiation of ES cells into cardiac myocytes. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induced acetylation of histone-3/4 near GATA sites within the atrial natriuretic factor promoter. In addition, TSA augmented the increase in an acetylated form of GATA-4 and its DNA binding during the ES cell differentiation. Finally, TSA facilitated the expression of green fluorescence protein under the control of the cardiac-specific Nkx-2.5 promoter and of endogenous cardiac beta-myosin heavy chain during the differentiation. These findings demonstrate that acetylation of GATA-4 as well as of histones is involved in the differentiation of ES cells into cardiac myocytes.

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

The differentiation of embryonic stem cells is associated with extensive changes in gene expression. It is not yet clear whether these changes are the result of binary switch-like mechanisms or that of continuous and progressive variation. Here, I have used immunostaining and single molecule RNA fluorescence in situ hybridization (FISH) to assess changes in the expression of the well-known pluripotency-associated gene Pou5f1 (also known as Oct4) and early differentiation markers Sox1 and T-brachyury in single cells during the early steps of differentiation of mouse embryonic stem cells. I found extensive overlap between the expression of Pou5f1/Sox1 or Pou5f1/T-brachyury shortly after the initiation of differentiation towards either the neuronal or the mesendodermal lineage, but no evidence of correlation between their respective expression levels. Quantitative analysis of transcriptional output at the sites of nascent transcription revealed that Pou5f1 and Sox1 were transcribed in pulses and that embryonic stem cell differentiation was accompanied by changes in pulsing frequencies. The progressive induction of Sox1 was further associated with an increase in the average size of individual transcriptional bursts. Surprisingly, single cells that actively and simultaneously transcribe both the pluripotency- and the lineage-associated genes could easily be found in the differentiating population. The results presented here show for the first time that lineage priming can occur in cells that are actively transcribing a pluripotent marker. Furthermore, they suggest that this process is associated with changes in transcriptional dynamics.

Does primordial germ cell (PGC) activation start before mouse embryo implantation, and does the possible regulation of the DNA (cytosine-5-)-methyltransferase 3-like (Dnmt3l) by transcription factor AP-2, gamma (TCFAP2C) have a role in this activation and in the primitive endoderm (PE)-epiblast (EPI) lineage specification? A burst of expression of PGC markers, such as Dppa3/Stella, Ifitm2/Fragilis, Fkbp6 and Prdm4, is observed from embryonic day (E) 3.25, and some of them, together with the late germ cell markers Zp3, Mcf2 and Morc1, become restricted to the EPI subpopulation at E4.5, while the dynamics analysis of the PE-EPI transitions in the single-cell data suggests that TCFAP2C transitorily represses Dnmt3l in EPI cells at E3.5 and such repression is withdrawn with reactivation of Dnmt3l expression in PE and EPI cells at E4.5. In the mouse preimplantation embryo, cells with the same phenotype take different fates based on the orchestration between topological clues (cell polarity, positional history and division orientation) and gene regulatory rules (at transcriptomics and epigenomics level), prompting the proposal of positional, stochastic and combined models explaining the specification mechanism. PGC specification starts at E6.0-6.5 post-implantation. In view of the important role of DNA methylation in developmental events, the cross-talk between some transcription factors and DNA methyltransferases is of particular relevance. TCFAP2C has a CpG DNA methylation motif that is not methylated in pluripotent cells and that could potentially bind on DNMT3L, the stimulatory DNA methyltransferase co-factor that assists in the process of de novo DNA methylation. Chromatin-immunoprecipitation analysis has demonstrated that Dnmt3l is indeed a target of TCFAP2C. We aimed to assess the timing of early preimplantation events and to understand better the segregation of the inner cell mass (ICM) into PE and EPI. We designed a single-cell transcriptomics dynamics computational study to identify markers of the PE-EPI bifurcation in ICM cells through searching for statistically significant (using the Student's t-test method) differently expressed genes (DEGs) between PE and EPI cells from E3.5 to E4.5. The DEGs common for E3.5 and E4.5 were used as the markers defining the steady states. We collected microarray and next-generation sequencing transcriptomics data from public databases from bulk populations and single cells from mice at E3.25, E3.5 and E4.5. The results are based on three independent single-cell transcriptomics data sets, with a fold change of 3 and P-value <0.01 for the DEG selection. The dynamics analysis revealed new transitory E3.5 and steady PE and EPI markers. Among the transitory E3.5 PE markers (Dnmt3l, Dusp4, Cpne8, Akap13, Dcaf12l1, Aaed1, B4galt6, BC100530, Rnpc3, Tfpi, Lgalsl, Ckap4 and Fbxl20), several (Dusp4, Akap13, Cpn8, Dcaf12l1 and Tfpi) are related to the extracellular regulated kinase pathway. We also identified new transitory E3.5 EPI markers (Sgk1, Mal, Ubxn2a, Atg16l2, Gm13102, Tcfap2c, Hexb, Slc1a1, Svip, Liph and Mier3), six new stable PE markers (Sdc4, Cpn1, Dkk1, Havcr1, F2r/Par1 and Slc7a6os) as well as three new stable EPI markers (Zp3, Mcf2 and Hexb), which are known to be late stage germ cell markers. We found that mouse PGC marker activation starts at least at E3.25 preimplantation. The transcriptomics dynamics analyses support the regulation of Dnmt3l expression by TCFAP2C. Since the regulation of Dnmt3l by TCFAP2C is based on computational prediction of DNA methylation motifs, Chip-Seq and transcriptomics data, functional studies are required to validate this result. We identified a collection of previously undescribed E3.5-specific PE and EPI markers, and new steady PE and EPI markers. Identification of these genes, many of which encode cell membrane proteins, will facilitate the isolation and characterization of early PE and EPI populations. Since it is so well established in the literature that mouse PGC specification is a post-implantation event, it was surprising for us to see activation of PGC markers as early as E3.25 preimplantation, and identify the newly found steady EPI markers as late germ cell markers. The discovery of such early activation of PGC markers has important implications in the derivation of germ cells from pluripotent cells (embryonic stem cells or induced pluripotent stem cells), since the initial stages of such derivation resemble early development. The early activation of PGC markers points out the difficulty of separating PGC cells from pluripotent populations. Collectively, our results suggest that the combining of the precision of single-cell omics data with dynamic analysis of time-series data can establish the timing of some developmental stages as earlier than previously thought. Not applicable. This work was supported by grants DFG15/14 and DFG15/020 from Diputación Foral de Gipuzkoa (Spain), and grant II14/00016 from I + D + I National Plan 2013-2016 (Spain) and FEDER funds. The authors declare no conflict of interest.

Miscarriages cause a greater loss-of-life than cardiovascular diseases, but knowledge about environmentally induced miscarriages is limited. Cultured naïve pluripotent embryonic stem cells (ESC) differentiate into extra-embryonic endoderm/extraembryonic endoderm (XEN) or formative pluripotent ESC, during the period emulating maximal miscarriage of peri-implantation development. In previous reports using small marker sets, hyperosmotic sorbitol, or retinoic acid (RA) decreased naïve pluripotency and increased XEN by FACS quantitation. Bulk and single cell (sc)RNAseq analyses of two cultured ESC lines was done, corroborated by qPCR. Transcriptomic responses were analyzed of cultured ESC stressed by Sorbitol, with Leukemia inhibitory factor (LIF + ; stemness growth factor), RA without LIF to control for XEN induction, and compared with normal differentiation (LIF - , ND). Sorbitol and RA increase subpopulations of 2-cell embryo-like (2CEL) and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE) cells and suppress formative pluripotency, imbalancing alternate lineage choices of initial naïve pluripotent cultured ESC compared with ND. Although bulk RNAseq and gene ontology (GO) group analyses suggest that stress induces anterior VE-head organizer and placental markers, scRNAseq reveals relatively few cells. But VE and placental markers/cells were in adjacent stressed cell clusters in the UMAP, like recent, normal UMAP of conceptuses. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance. Hyperosmotic stress, and other toxicological stresses, like drugs with active ingredient RA, may cause premature, lineage imbalance, resulting in miscarriages or birth defects.

Hepatic differentiation of human embryonic stem cells by coupling substrate stiffness and microtopography.