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Directed in vitro differentiation of pluripotent stem cells toward definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two-dimensional monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (

The intestine and colon carries out vital functions, and their lifelong maintenance is of the upmost importance. Research over the past decades has carefully addressed bowel function, how it is maintained and begun to unravel how disorders such as cancer and inflammatory bowel disease form. In contrast, very little is known about the molecular mechanisms that trigger tissue maturation during development. With this review, our aim is to carefully provide a critical appraisal of the literature to give a state-of-the-art view of intestinal development. Starting from definitive endoderm at gastrulation to the emergence of a structure with mature properties, the tissue undergoes complex morphogenetic processes that rely on both biophysical changes and secreted signaling molecules. We will also discuss how new and exciting developments using in vitro models are likely to provide new insights into this process and potential therapeutic strategies for gastrointestinal disorders.

We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1α and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1α(-/-) mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1α(-/-) mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1α(-/-) mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.

Dynamic suspension culture of human pluripotent stem cells (hPSCs) in stirred bioreactors provides a valuable scalable culture platform for integrated differentiation toward different lineages for potential research and therapeutic applications. However, current protocols for scalable and integrated differentiation of hPSCs limited due to high cost of growth factors and technical challenges. Here, hPSCs aggregates primed with 6 and 12 μM of CHIR99021 (CHIR), a Wnt agonist, in combination with different concentrations of high cost Activin A (10, 25, 50, 100 ng/mL). We sought to determine the appropriate treatment duration for efficient and cost-effective differentiation protocol for foregut definitive endoderm production in a dynamic suspension culture. Afterward, we evaluated the impact of the initial hPSC aggregate sizes (small: 86 ± 18 μm; medium: 142 ± 32 μm; large: 214 ± 34 μm) as critical bioprocess parameter on differentiation efficacy at the beginning of induction. The results indicated that 1-day priming of hPSCs as 3D aggregates (hPSpheres) with 6 μM CHIR followed by treatment with a low concentration of Activin (10 ng/mL) for 2 days resulted in efficient differentiation to definitive endoderm. This finding confirmed by the presence of ≥70% SOX17/FOXA2-double positive cells that highly expressed the anterior endodermal marker HEX. These endodermal cells differentiated efficiently into mature functional hepatocytes [60% albumin (ALB)-positive cells]. The results showed that the initial size of hPSC aggregates significantly impacted on the efficacy of differentiation. The medium sized-hPSpheres resulted in higher productivity and differentiation efficiency for scalable hepatocytes production, whereas small aggregates resulted in significant cell-loss after CHIR treatment and large aggregates had less efficacious endodermal differentiation. Differentiated cells exhibited multiple characteristics of primary hepatocytes as evidenced by expressions of liver-specific markers, indocyanine green and low-density lipoprotein uptake, and glycogen storage. Thus, this platform could be employed for scalable production of hPSC-derived hepatocytes for clinical and drug discovery applications.

Differentiation of pluripotent cells into endoderm-related cell types initially requires in vitro gastrulation into the definitive endoderm (DE). Most differentiation protocols are initiated from colonies of pluripotent cells complicating their adaption due to insufficiently defined starting conditions. The protocol described here was initiated from a defined cell number of dispersed single cells and tested on three different human embryonic stem cell lines and one human induced pluripotent stem cell line. Combined activation of ActivinA/Nodal signaling and GSK3 inhibition for the first 24 h, followed by ActivinA/Nodal signaling efficiently induced the DE state. Activation of ActivinA/Nodal signaling alone was not effective. Efficient GSK3 inhibition allowed the reduction of the ActivinA concentration during the entire protocol. A feeder-independent cultivation of pluripotent cells was preferred to achieve the high efficiency and robustness since feeder cells hindered the differentiation process. Additionally, inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway was not required, nonetheless yielding high cell numbers efficiently committed toward the DE. Finally, the endoderm generated could be differentiated further into PDX1-positive pan-pancreatic cells and NGN3-positive endocrine progenitors. Thus, this efficient and robust DE differentiation protocol is a step forward toward better reproducibility due to the well-defined conditions based on dispersed single cells from feeder-free-cultivated human pluripotent cells.

Pancreatic progenitor (PP) cells are tissue-committed cells, which can differentiate into all kinds of pancreatic cells. They are potential candidates for regeneration of pancreatic tissue. However, it is unfeasible to acquire PP cells from pancreatic tissues and expand them in vitro. Generation of PP cells from adipose tissue-derived mesenchymal stem cells (AD-MSCs) would provide an unlimited source of PP cells. Here we developed a 2-step stepwise protocol, which induced AD-MSCs to generate FOXA2- or SOX17-positive definitive endoderm (DE) (5 days) and pancreatic and duodenal homeobox gene 1 (PDX1)-positive PP cells (4-6 days). By mimicking the developmental progress in embryonic development, we optimized the timing and combination of cytokines to activate the key signaling pathways during pancreatic development. We found that activating the Nodal/Activin signal with Activin A could induce differentiation of AD-MSCs toward DE, which could be further promoted by the Wnt signaling pathway activator Wnt3a. Besides, transient T (BRACHYURY)(+) mesendodermal cells were observed during formation of DE from AD-MSCs. Subsequently, the Wnt signaling pathway inhibitor Dkk1 along with retinoic acid/FGF2 (60 ng/mL) further induced AD-MSC-derived DE cells to differentiate into PDX1-positive PP cells. The derived PP cells were capable to form pancreatic endocrine or exocrine cells. In conclusion, we established a stepwise protocol that could derive DE and PP cells from AD-MSCs. It might provide an unlimited source of autologous PP cells for pancreatic diseases.

Despite the enormous progress in studying definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs), none of the reported protocols have produced a universal, cost-effective, and competent DE with the capability to further differentiate into endodermal derivatives. In this study, by using a 2-step differentiation strategy, we have treated hESCs for 1 day with "priming" small molecules (SM), [stauprimide, NSC-308848, rapamycin (Rapa), and/or CHIR] and for the next 3 days with "inducing" SM (LY294002, cymarin, IDE1, and/or IDE2) in conjunction with activin A. In the positive control group, we treated hESCs with Wnt3a (25 ng/mL) for 1 day and activin A (100 ng/mL; W/A100-A100) for the next 3 days. Gene expression analysis showed that treatment of hESCs with 100 nM Rapa and 50 ng/mL activin A (Rapa-A50) out of 25 combinations of factors gave rise to higher expressions of 2 DE-specific genes, SOX17 and FOXA2. Similar results were obtained after treating 2 other hESC lines with this regimen. To investigate the competency of Rapa-A50-induced DE for further differentiation into endodermal derivatives, these cells and W/A100-A100-induced DE cells (positive control) were further differentiated into pancreatic progenitors (PP), then into pancreatic endocrine (PE) cells using 5 previously described differentiation protocols. Gene analysis of differentiated cells showed that the established protocols were insufficient to enable universal differentiation into PE, whereas Rapa-A50-induced DE cells were more competent for PP differentiation in a protocol-dependent manner. Additionally, Rapa-A50-induced DE had the capability to differentiate into hepatocyte-like cells (HLCs) as efficiently as W/A100-A100-induced DE. These data have indicated that hESCs primed with Rapa, and induced by a lower concentration of activin A, could lead to DE that had the capability to further differentiate into HLCs and PP cells, but not PE cells. Thus, current protocols for the differentiation of DE into PE still need additional study.

Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as liver, pancreas, and intestine. However, purification of DE in mammals has not been achieved, limiting the molecular "definition" of endoderm, and hindering our understanding of DE development and attempts to produce endoderm from sources such as embryonic stem (ES) cells. Here, we describe purification of mouse DE using fluorescence-activated cell sorting (FACS) and mice harboring a transgene encoding enhanced green fluorescent protein (eGFP) inserted into the Sox17 locus, which is expressed in the embryonic endoderm. Comparison of patterns of signaling pathway activation in native mouse DE and endoderm-like cells generated from ES cells produced novel culture modifications that generated Sox17-eGFP⁺ progeny whose gene expression resembled DE more closely than achieved with standard methods. These studies also produced new FACS methods for purifying DE from nontransgenic mice and mouse ES cell cultures. Parallel studies of a new human SOX17-eGFP ES cell line allowed analysis of endoderm differentiation in vitro, leading to culture modifications that enhanced expression of an endoderm-like signature. This work should accelerate our understanding of mechanisms regulating DE development in mice and humans, and guide further use of ES cells for tissue replacement.

Cultivation of functional pancreatic cells isolated from adult mammalian pancreas remains difficult. We developed a differentiation protocol that gradually induced the formation of mouse pancreatic exocrine cells from embryonic stem cells (ESCs). This process mimicked in vivo pancreatic development by directing cells through definitive endoderm (DE), gut tube endoderm, and pancreatic progenitor cells to differentiated cells that expressed pancreatic exocrine enzymes. Mouse ESCs were cultured in hanging drops to form embryoid bodies. Treatment of embryoid bodies with activin A induced the formation of DE cells that expressed marker mRNAs Goosecoid and Mixl1 and that were double-positive with Foxa2 and Sox17 proteins. Subsequent treatment of the DE cells by retinoic acid induced the formation of gut tube endoderm cells that expressed the specific marker Hnf1b. Expression of Goosecoid and Mixl1 was downregulated during this period. Fibroblast growth factor 7 (FGF7) promoted differentiation of PDX1-expressing pancreatic progenitor cells that also expressed Foxa2 mRNA, an endodermal marker, suggesting derivation from the DE cells. Exocrine cell differentiation was induced with FGF7, glucagon-like peptide-1, and nicotinamide. The differentiated cells expressed mature pancreatic exocrine cell mRNAs, such as Amylase, Elastase, and Carboxypeptidase A. Additionally, they produced pancreatic elastase, amylase, carboxypeptidase A, and chymotrypsin proteins that were identified in cytoplasmic granules by immunocytochemistry. Active amylase was released into the medium. Moreover, FGF7 was associated with differentiation of pancreatic exocrine cells. The findings reported here offer a novel and effective process to develop pancreatic exocrine cells from ESCs.

Mouse embryonic fibroblasts (MEFs) have been extensively used as feeder cells to support the in vitro propagation of human embryonic stem cells (hESCs). However, owing to the risk of cross-contamination with animal or other unknown pathogens, the use of MEFs does not meet requirements for the clinical application of hESCs. Moreover, the actual role played by the feeders in the differentiation of hESCs is still unclear. In this study, human embryonic fibroblasts (HEFs) were used as feeder cells to support the establishment and undifferentiated growth of hESCs, and the capability of HEFs to induce the differentiation of definitive endoderm (DE) was evaluated. Three new hES cell lines were derived. These cell lines exhibited and maintained the common features of traditional hESCs after prolonged culture in vitro. Furthermore, DE differentiation of the newly established hES cell lines was performed using 100 ng/ml activin A, and the effects were compared among HEFs, MEFs, and feeder-free systems. On day 5 of induction, DE (SOX17(+)) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0 +/- 8.9% and 78.7 +/- 3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7 +/- 5.6%). The SOX17(+) cells tended to differentiate into an endodermal lineage in vivo and could be further induced into glucagon and C-peptide double positive islet-like clusters in vitro. Our studies suggest that, in terms of therapeutic application, HEFs can be an effective substitute for MEFs for sustaining the derivation and DE differentiation of hESCs.

We previously reported that chick anterolateral endoderm (AL endoderm) induces cardiomyogenesis in mouse embryoid bodies. However, the requirement to micro-dissect AL endoderm from gastrulation-stage embryos precludes its use to identify novel cardiomyogenic factors, or to scale up cardiomyocyte numbers for therapeutic experiments. To circumvent this problem we have addressed whether human definitive endoderm (hDE) cells, which can be efficiently generated in large numbers from human embryonic stem cells (hESCs), can mimic the ability of AL endoderm to induce cardiac myogenesis. Results demonstrate that both hDE cells and medium conditioned by them induce cardiac myogenesis in pluripotent hESCs, as indicated by rhythmic beating and immunohistochemical/quantitative polymerase chain reaction monitoring of marker gene expression. The cardiomyogenic effect of hDE is enhanced when pluripotent hESCs are preinduced to the mes-endoderm state. Because this approach is tractable and scalable, it may facilitate identification of novel hDE-secreted factors for inclusion in defined cardiomyogenic cocktails.

Human embryonic stem cells (hESCs) have the potential to differentiate into many adult cell types, and they are being explored as a resource for cell replacement therapies for multiple diseases. In order to optimize in vitro differentiation protocols, it will be necessary to elucidate regulatory mechanisms that contribute to lineage specification. MicroRNAs (miRNAs) are emerging as key regulators of hESC differentiation and embryonic development. In this study, we compare miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE), an early step in the pathway toward the pancreatic lineage. Results from microarray analysis showed that DE can be distinguished by its unique miRNA profile, which consists of 37 significantly down-regulated and 17 up-regulated miRNAs in 2 different cell lines and in the presence/absence of feeder layers. Comparison to other hESC-derived lineages showed that most of the highly up-regulated miRNAs are specific to endoderm in early development. Notably, miR-375, which was previously implicated in regulating development and function of later stages of pancreatic development, is highly and specifically up-regulated during DE formation, suggesting that it may have a distinct role very early in development. Examination of potential mRNA targets showed that TIMM8A is repressed by ectopic miR-375 expression in pluripotent hESCs.

Human embryonic stem (hES) cells cultured in suspension form aggregates called embryoid bodies (EBs), which structurally resemble the pregastrulation-stage embryo. We show that these EBs express genes characteristic of the visceral endoderm (VE) and form an outer boundary of VE-like cells, but only a minority expresses markers of the primitive streak and the definitive endoderm in patterns suggestive of gastrulation-like events. EBs embedded in a Matrigel matrix, however, lack the presumptive VE but up-regulate expression of gastrulation-related genes, which is correlated with the expression of these genes in a greater proportion of individual EBs. Over time, Matrigelembedded EBs form internally organized structures and exhibit higher expression of markers of the definitive endoderm and mesoderm. We hypothesize that a three-dimensional adhesive support provides an organizing influence analogous to the early basal lamina surrounding the epiblast of the pregastrulation embryo. Furthermore, we identify a synthetic three-dimensional scaffold capable of supporting EB formation that partially mimics the effects of Matrigel.