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Pluripotent stem cells (PSCs) hold enormous potential for treating multiple diseases owing to their ability to self‐renew and differentiate into any cell type. Albeit possessing such promising potential, controlling their differentiation into a desired cell type continues to be a challenge. Recent studies suggest that PSCs respond to different substrate stiffness and, therefore, can differentiate towards some lineages via Hippo pathway. Human PSCs can also differentiate and self‐organize into functional cells, such as organoids. Traditionally, human PSCs are differentiated on stiff plastic or glass plates towards definitive endoderm and then into functional pancreatic progenitor cells in the presence of soluble growth factors. Thus, whether stiffness plays any role in differentiation towards definitive endoderm from human pluripotent stem cells (hPSCs) remains unclear. Our study found that the directed differentiation of human embryonic stem cells towards endodermal lineage on the varying stiffness did not differ from the differentiation on stiff plastic dishes. We also observed no statistical difference between the expression of yes‐associated protein (YAP) and phosphorylated YAP. Furthermore, we demonstrate that lysophosphatidic acid, a YAP activator, enhanced definitive endoderm formation, whereas verteporfin, a YAP inhibitor, did not have the significant effect on the differentiation. In summary, our results suggest that human embryonic stem cells may not differentiate in response to changes in stiffness, and that such cues may not have as significant impact on the level of YAP. Our findings indicate that more research is needed to understand the direct relationship between biophysical forces and hPSCs differentiation. [ABSTRACT FROM AUTHOR]

With self‐renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain‐containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3‐dependent transcriptomic changes during endoderm differentiation of mESCs using time‐course RNA‐seq analysis. We found that SETD3 is involved in the timely activation of the endoderm‐related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3‐deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF β‐catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and β‐catenin. We were able to rescue the differentiation defect by stably re‐expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of β‐catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs. [ABSTRACT FROM AUTHOR]

Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (ActivinAandWnt-3a) and smallmolecules (sodiumbutyrate and IDE1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapidDE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications.We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications. [ABSTRACT FROM AUTHOR]

Derivation of definitive endoderm (DE) from human embryonic stem cells (hESCs) can address the needs of regenerative medicine for endoderm-derived organs such as the pancreas and liver. Fibrous substrates which topographically recapitulate native extracellular matrix have been known to promote the stem cell differentiation. However, the optimal fiber diameter remains to be determined for the desired differentiation. Here, we have developed a simple method to precisely fabricate electrospun poly(ε-caprolactone) fibers with four distinct average diameters at nano- and microscale levels (200, 500, 800, and 1300 nm). Human ESCs were cultured as clumps or single cells and induced into DE differentiation to determine the optimal topography leading to the promoted differentiation compared with planar culture plates. Gene expression analysis of the DE-induced cells showed significant upregulation of DE-specific genes exclusively on the 200-nm fibers. By Western blot analysis, significant expression of DE-specific proteins was found when hESCs were cultured on the 200 nm substrate as single cells rather than clumps, probably due to more efficient cell-matrix interaction realized by morphological observations of the cell colonies. The results indicated that nanofibrillar substrates, only at ultrathin fiber diameters, provided a better environment for DE differentiation of hESC, which holds great promise in prospective tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3539-3553, 2015. [ABSTRACT FROM AUTHOR]

The first step in the formation of hepatocytes and beta cells is the generation of definitive endoderm (DE) which involves a central issue in developmental biology. Human induced pluripotent stem cells (hiPSCs) have the pluripotency to differentiate into all three germ layers in vitro and have been considered potent candidates for regenerative medicine as an unlimited source of cells for therapeutic applications. In this study, we investigated the differentiating potential of hiPSCs on poly (ε-caprolactone) (PCL) nanofibrous scaffold into DE cells. Here, we demonstrate directed differentiation of hiPSCs by factors such as Activin A and Wnt3a. The differentiation was determined by immunofluoresence staining with Sox17, FoxA2 and Goosecoid (Gsc) and also by qRT-PCR analysis. The results of this study showed that hiPSCs, as a new cell source, have the ability to differentiate into DE cells with a high capacity and also demonstrate that three dimension (3D) culture provides a suitable nanoenviroment for growth, proliferation and differentiation of hiPSCs. PCL nanofibrous scaffold with essential supplements, stimulating factors and EB-derived cells is able to provide a novel method for enhancing functional differentiation of hiPSCs into DE cells. [ABSTRACT FROM AUTHOR]

Human-induced pluripotent stem cells (hiPSCs) are considered to be potentially able to differentiate into all human cell lineages and thus hold promise as an unlimited source for cell replacement therapies in clinical applications. Definitive endoderm (DE) formation is the first and crucial step in the development of visceral organs such as liver, lung, pancreas and so forth. Therefore, efficient generation of DE cells ensures the efficient generation of eventual target cells used in cell therapy. In the present study, Matrigel-coated poly(lactic acid)/gelatin (PLA/gelatin) nanofibrous scaffolds were utilized to investigate the proliferation and differentiation of hiPSCs into DE cells. Analyses of DE-specific markers including Sox17, FoxA2, and Gooscoid (Gsc) genes revealed higher levels of mRNA and protein expression in the differentiated hiPSCs cells cultured on PLA/gelatin scaffolds than cells differentiated in two-dimensional (2D) culture. Our results showed that three-dimensional (3D) cultures could significantly promote DE differentiation in comparison with 2D culture. Also using small molecules such as inducer of definitive endoderm 1 (IDE1) and signaling molecules such as Activin A and Wnt3a could enhance the DE differentiation of hiPSCs with Activin A/Wnt3a being significantly more potent in both 2D and 3D cultures compared to IDE1. The results of this study may have impact in tissue engineering and cell replacement therapy of visceral organs-related diseases. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 4027-4036, 2014. [ABSTRACT FROM AUTHOR]

Aim Hepatic non-parenchymal cells are well known to be capable of providing an important microenvironment and growth factors for hepatic regeneration, but their capacity for directing embryonic stem cells ( ESC) toward hepatocytes remains to be assessed. Thus, this study aims to investigate the role of hepatic stellate cells ( HSC), the major type of hepatic non-parenchymal cells, in the differentiation of ESC as well as exploring the potentiality of ESC in regeneration medicine for cell-based therapy. Methods A two-step differentiation procedure that utilized the capability of HSC to regulate proliferation and differentiation of hepatocytes was used to develop an approach for directing the differentiation of ESC towards hepatic progenitor cells. Mouse ESC were cultivated in a serum-free medium containing Activin A and fibroblast growth factor to generate definitive endodermal cells characterized by the CXCR4 cell-surface marker. After 6-8 days in culture, approximately 60% of the differentiated cells expressed CXCR4, and more than 90% of the CXCR4 positive cells could be recovered by cell sorting. The purified CXCR4 positive cells were co-cultured with mouse HSC as feeder cells in basal medium without additional hepatocyte growth factors. Differentiation was complete after 10-12 days of co-culture, and hepatic progenitor cell markers such as α-fetoprotein ( afp) and albumin ( alb) were detected in the terminally differentiated ESC. Conclusion These results show that HSC provide an appropriate microenvironment and pivotal growth factors for generation of hepatic progenitor cells from ESC-derived definitive endodermal cells, and suggest that this approach possibly allows for hepatic differentiation of ESC imitating the process of hepatic regeneration. [ABSTRACT FROM AUTHOR]

In recent years the multipotent extraembryonic endoderm ( XEN) stem cells have been the center of much attention. In vivo, XEN cells contribute to the formation of the extraembryonic endoderm, visceral and parietal endoderm and later on, the yolk sac. Recent data have shown that the distinction between embryonic and extraembryonic endoderm is not as strict as previously thought due to the integration, and not the displacement, of the visceral endoderm into the definitive embryonic endoderm. Therefore, cells from the extraembryonic endoderm also contribute to definitive endoderm. Many research groups focused on unraveling the potential and ability of XEN cells to both support differentiation and/or differentiate into endoderm-like tissues as an alternative to embryonic stem ( ES) cells. Moreover, the conversion of ES to XEN cells, shown recently without genetic manipulations, uncovers significant and novel molecular mechanisms involved in extraembryonic endoderm and definitive endoderm development. XEN cell lines provide a unique model for an early mammalian lineage that complements the established ES and trophoblast stem cell lines. Through the study of essential genes and signaling requirements for XEN cells in vitro, insights will be gained about the developmental program of the extraembryonic and embryonic endodermal lineage in vivo. This review will provide an overview on the current literature focusing on XEN cells as a model for primitive endoderm and possibly definitive endoderm as well as the potential of using these cells for therapeutic applications. [ABSTRACT FROM AUTHOR]

The Sox family of HMG (high mobility group)-box transcription factors are highly conserved in vertebrates. Sox members are involved in various developmental processes. Among them Sox17 has been demonstrated to function as an endoderm determinant in zebrafish and Xenopus, respectively. However, little is known about the role of Sox17 in mouse embryonic stem cell (ESC) differentiation. In our research, we investigated the effect of Sox17 on mouse ESC and embryoid body (EB) differentiation. The results demonstrated that Sox17 overexpression upregulated a set of endoderm-specific gene markers, suggesting that Sox17 overexpression induced an ESC differentiation program towards both primitive and definitive endoderm. We believe this finding brings new insights into the understanding of ESC differentiation and the organogenesis of endodermal derivatives. [ABSTRACT FROM AUTHOR]