Analysis of gene expressions of embryonic stem‐derived Pdx1‐expressing cells: Implications of genes involved in pancreas differentiation

We have recently reported the method by which embryonic stem (ES) cells were induced into Pdx1‐expressing cells. To gain insights into the ES cell‐derived Pdx1‐expressing cells, we examined gene expression profiles of the cells by microarray experiments. Microarray analyses followed by a comparison with the data of the cells in developing pancreatic and adult islet suggested that the ES cell‐derived Pdx1‐positive cells were immature pancreatic progenitor cells with endodermal characteristics. The analyses of the genes upregulated in the ES cell‐derived Pdx1‐positive cells would give us knowledge on early pancreatic development. Here, we first listed the genes and found that these contained not only those known to be expressed in the endoderm or pancreatic progenitor cells, but also those known to be involved in left–right axis formation. Second, we examined the gene expression patterns and found that several genes were expressed in the ventral foregut lip at the anterior intestinal portal in E8.5 embryo. Given that the Pdx1/GFP‐expressing cells are first observed in the same region at the anterior intestinal portal, these results suggest that the pancreatic progenitor cells first give rise at the ventral endoderm prior to the formation of dorsal and ventral pancreatic buds.     

Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors acquire the character of XEN cells

Background  

Three types of cell lines have been established from mouse blastocysts: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extra-embryonic endoderm (XEN) cells, which have the potential to differentiate into their respective cognate lineages. ES cells can differentiate in vitro not only into somatic cell lineages but into extra-embryonic lineages, including trophectoderm and extra-embryonic endoderm (ExEn) as well. TS cells can be established from ES cells by the artificial repression of Oct3/4 or the upregulation of Cdx2 in the presence of FGF4 on feeder cells. The relationship between these embryo-derived XEN cells and ES cell-derived ExEn cell lines remains unclear, although we have previously reported that overexpression of Gata4 or Gata6 induces differentiation of mouse ES cells into extra-embryonic endoderm in vitro.     

Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells

Background  

Rex1/Zfp42 has been extensively used as a marker for the undifferentiated state of pluripotent stem cells. However, its function in pluripotent stem cells including embryonic stem (ES) cells remained unclear although its involvement in visceral endoderm differentiation in F9 embryonal carcinoma (EC) cells was reported.     

The identity and distribution of neural cells expressing the mesodermal determinant spadetail

Background  

The spadetail (spt) gene of zebrafish is expressed in presomitic mesoderm and in neural cells previously suggested to be Rohon-Beard neurons. The mechanism(s) generating the apparently irregular rostrocaudal distribution of spt-expressing cells in the developing CNS is unknown.     

Gene expression profiles during early differentiation of mouse embryonic stem cells

Background  

Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1–4 EBs     

Multiple mesodermal lineage differentiation of Apodemus sylvaticus embryonic stem cells in vitro

Background

Embryonic stem (ES) cells have attracted significant attention from researchers around the world because of their ability to undergo indefinite self-renewal and produce derivatives from the three cell lineages, which has enormous value in research and clinical applications. Until now, many ES cell lines of different mammals have been established and studied. In addition, recently, AS-ES1 cells derived from Apodemus sylvaticus were established and identified by our laboratory as a new mammalian ES cell line. Hence further research, in the application of AS-ES1 cells, is warranted.

Results

Herein we report the generation of multiple mesodermal AS-ES1 lineages via embryoid body (EB) formation by the hanging drop method and the addition of particular reagents and factors for induction at the stage of EB attachment. The AS-ES1 cells generated separately in vitro included: adipocytes, osteoblasts, chondrocytes and cardiomyocytes. Histochemical staining, immunofluorescent staining and RT-PCR were carried out to confirm the formation of multiple mesodermal lineage cells.

Conclusions

The appropriate reagents and culture milieu used in mesodermal differentiation of mouse ES cells also guide the differentiation of in vitro AS-ES1 cells into distinct mesoderm-derived cells. This study provides a better understanding of the characteristics of AS-ES1 cells, a new species ES cell line and promotes the use of Apodemus ES cells as a complement to mouse ES cells in future studies.     

Global expression of cell surface proteins in embryonic stem cells

Background

Recent studies have shown that embryonic stem (ES) cells globally express most genes in the genome at the mRNA level; however, it is unclear whether this global expression is propagated to the protein level. Cell surface proteins could perform critical functions in ES cells, so determining whether ES cells globally express cell surface proteins would have significant implications for ES cell biology.

Methods and Principal Findings

The surface proteins of mouse ES cells were purified by biotin labeling and subjected to proteomics analysis. About 1000 transmembrane or secreted cell surface proteins were identified. These proteins covered a large variety if functional categories including signal transduction, adhesion and transporting. More over, mES cells promiscuously expressed a wide variety of tissue specific surface proteins. And many surface proteins were expressed heterogeneously on mES cells. We also find that human ES cells express a wide variety of tissue specific surface proteins.

Conclusions/Significance

Our results indicate that global gene expression is not simply a result of leaky gene expression, which could be attributed to the loose chromatin structure of ES cells; it is also propagated to the functional level. ES cells may use diverse surface proteins to receive signals from the diverse extracellular stimuli that initiate differentiation. Moreover, the promiscuous expression of tissue specific surface proteins illuminate new insights into the strategies of cell surface marker screening.     

Identification and targeted disruption of the mouse gene encoding ESG1 (PH34/ECAT2/DPPA5)

Background  

Embryonic stem cell-specific gene (ESG) 1, which encodes a KH-domain containing protein, is specifically expressed in early embryos, germ cells, and embryonic stem (ES) cells. Previous studies identified genomic clones containing the mouse ESG1 gene and five pseudogenes. However, their chromosomal localizations or physiological functions have not been determined.     

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17⁺ endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17⁺ cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP⁺ cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17⁺ cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.     

Differential genomic imprinting and expression of imprinted microRNAs in testes-derived male germ-line stem cells in mouse

Background

Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. Consequently, a molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings.

Methods and Findings

Using mouse as a model system, here we show that, similar to sperm, expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) were consistently higher (P<0.001), while those of imprinted and maternally expressed miRNA (miR-127, miR-127-5p) were consistently lower (P<0.001) in GS cells than in control embryonic stem (ES) cells. DNA methylation analyses of imprinting control regions (ICR), that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR), confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. The expression of imprinted miRNAs in GS and maGS cells were also altered during their in vitro differentiation and varied both with the differentiation stage and the miRNA.

Conclusions

Our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus, serve as epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells.     

FoxO1 inhibition promotes differentiation of human embryonic stem cells into insulin producing cells

Insulin-producing cells (IPCs) derived from human embryonic stem cells (hESCs) hold great potential for cell transplantation therapy in diabetes. Tremendous progress has been made in inducing differentiation of hESCs into IPCs in vitro, of which definitive endoderm (DE) protocol mimicking foetal pancreatic development has been widely used. However, immaturity of the obtained IPCs limits their further applications in treating diabetes. Forkhead box O1 (FoxO1) is involved in the differentiation and functional maintenance of murine pancreatic β cells, but its role in human β cell differentiation is under elucidation. Here, we showed that although FoxO1 expression level remained consistent, cytoplasmic phosphorylated FoxO1 protein level increased during IPC differentiation of hESCs induced by DE protocol. Lentiviral silencing of FoxO1 in pancreatic progenitors upregulated the levels of pancreatic islet differentiation-related genes and improved glucose-stimulated insulin secretion response in their progeny IPCs, whereas overexpression of FoxO1 showed the opposite effects. Notably, treatment with the FoxO1 inhibitor AS1842856 displayed similar effects with FoxO1 knockdown in pancreatic progenitors. These effects were closely associated with the mutually exclusive nucleocytoplasmic shuttling of FoxO1 and Pdx1 in the AS1842856-treated pancreatic progenitors. Our data demonstrated a promising effect of FoxO1 inhibition by the small molecule on gene expression profile during the differentiation, and in turn, on determining IPC maturation via modulating subcellular location of FoxO1 and Pdx1. Therefore, we identify a novel role of FoxO1 inhibition in promoting IPC differentiation of hESCs, which may provide clues for induction of mature β cells from hESCs and clinical applications in regenerative medicine.