Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.     

Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

Embryoid bodies (EBs) are primitive embryonic structures derived from differentiating embryonic stem cells (ESCs). Many techniques have been used to obtain EBs. Improving the technique of EB formation can help in achieving better results in ESCs differentiation into neurons, myocardiocytes, haemopoeitic cells, and others. We evaluated the use of Sigmacote™ as a hydrophobic substrate to improve EB formation. CCE and P19 cell lines were used to obtain EBs and retinoic acid was used to induce neural differentiation. The results revealed that Sigmacote™, as a hydrophobic substrate, can improve EB formation from ESCs. Our results demonstrate that the silicon-coating of glass petri dishes by Sigmacote™ is an easy and reproducible technique to enhance EB formation from murine ESCs and EC cells.     

Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies

The mammalian reproductive tract is known to contain 1.5–5.3% oxygen (O₂), but human embryonic stem cells (hESCs) derived from preimplantation embryos are typically cultured under 21% O₂ tension. The aim of this study was to investigate the effects of O₂ tension on the long-term culture of hESCs and on cell-fate determination during early differentiation. hESCs and embryoid bodies (EBs) were grown under different O₂ tensions (3, 12, and 21% O₂). The expression of markers associated with pluripotency, embryonic germ layers, and hypoxia was analyzed using RTPCR, immunostaining, and Western blotting. Proliferation, apoptosis, and chromosomal aberrations were examined using BrdU incorporation, caspase-3 immunostaining, and karyotype analysis, respectively. Structural and morphological changes of EBs under different O₂ tensions were comparatively examined using azan- and hematoxylineosin staining, and scanning and transmission electron microscopy. Mild hypoxia (12% O₂) increased the number of cells expressing Oct4/Nanog and reduced BrdU incorporation and aneuploidy. The percentage of cells positive for active caspase-3, which was high during normoxia (21% O₂), gradually decreased when hESCs were continuously cultured under mild hypoxia. EBs subjected to hypoxia (3% O₂) exhibited well-differentiated microvilli on their surface, secreted high levels of collagen, and showed enhanced differentiation into primitive endoderm. These changes were associated with increased expression of Foxa2, Sox17, AFP, and GATA4 on the EB periphery. Our data suggest that mild hypoxia facilitates the slow mitotic division of hESCs in long-term culture and reduces the frequency of chromosomal abnormalities and apoptosis. In addition, hypoxia promotes the differentiation of EBs into extraembryonic endoderm.     

Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies

Retinoid signaling has been implicated in embryonic stem cell differentiation. Here we present a systematic analysis of gene expression changes in mouse embryonic stem cells (mESCs), during their spontaneous differentiation into embryoid bodies and the effect of all-trans retinoic acid (ATRA) on this process. We show that retinoic acid is present in the serum and is sufficient to activate retinoid signaling at a basal level in undifferentiated mESCs. This signal disappears during embryoid body formation. However exogenously added ATRA resets the spontaneous differentiation programs in embryoid bodies and initiates a distinct genetic program. These data suggest that retinoid signaling not only promotes a particular pathway but also acts as a context dependent general coordinator of the differentiation states in embryonic stem cells.     

Physical passaging of embryoid bodies generated from human pluripotent stem cells

Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1∶4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells.     

In vitro differentiation of embryonic stem cells: Immunophenotypic analysis of cultured embryoid bodies

The process of in vitro embryonic stem cell differentiation and embryoid body development was monitored using a panel of antibodies against surface markers traditionally associated with embryonic tissue (Forssman, SSEA-1) and hematopoietic progenitor cells (Fall-3, HSA, Sca-1, Thy-1.2, ER-MP12, CD45, AA4.1, and c-kit). All markers with the exception of CD45 and AA4.1 were initially detected in cultures of undifferentiated ES cells. During the first 11 days of differentiation, distinct and reproducible patterns of surface expression were observed for each marker. Using the kinetic display of surface markers as a gauge of differentiation, perturbations in embryoid body development were detected in cultures supplemented with interleukin-11, a gp130-activating cytokine thought to affect embryonic stem cell differentiation. In the absence of exogenous cytokines, microbead immunoselected day 7 c-kit, ER-MP12, and CD45-positive embryoid body cells were enriched for hematopoietic progenitors as detected by methylcellulose colony assays, while no significant enrichment of hematopoietic progenitors was observed with Sca-1, Thy-1.2, Fall-3, and Forssman-immunoselected cells. These results indicate that the process of early embryoid body development is associated with a programmed sequence of cell surface marker display, concomitant with the development of phenotypically definable embryonic cell lineages. J. Cell. Physiol. 171:104–115, 1997. © 1997 Wiley-Liss, Inc.     

Temporal changes in the carbohydrates expressed on BG01 human embryonic stem cells during differentiation as embryoid bodies

Cell surface carbohydrates present on BG01 human embryonic stem cells after 28 days of differentiation were examined using two classes of carbohydrate binding proteins: lectins and antibodies specific for carbohydrate epitopes. Specificity of lectin staining was verified using carbohydrate ligands to block lectin interaction, glycohydrolases to cleave specific sugar residues that are receptors for these proteins, and periodate oxidation to destroy susceptible sugar residues. Specific antibodies were used to identify various tissue types and germ layers present in the 12- and 28-day differentiating embryoid bodies. Results from 12 and 28-day differentiated embryoid bodies were compared to determine changes over time. A slight increase in the sialylation of α-GalNAc was seen between 12 and 28 days of differentiation due to the presence of sialyl Tn and/or other sialylated α-GalNAc residues. Increases were also observed in GalNAc, the T antigen (Gal β1,3 GalNAc), and difucosylated LacNAc residues during this time interval. Additionally, some distinct differences in the pattern of lectin staining between 12 and 28 days were observed. Not unexpectedly, the presence of most differentiated cell-types increased during this time period with the exception of neural progenitors, which decreased. Undifferentiated cells, which were prevalent in the 12-day EBs, were undetectable after 28 days. We conclude that several changes in glycosylation occurred during the differentiation of embryonic stem cells, and that these changes may play a role in embryonic development. Lectin abbreviations can be found in Table 1.     

Activin B mediated induction of Pdx1 in human embryonic stem cell derived embryoid bodies

Human embryonic stem cells (hESCs) have the potential to provide alternative sources for pancreatic islet grafts. In the present study we have investigated the influence of Activin A and Activin B on the expression of the pancreas marker gene Pdx1 in hESCs differentiated as embryoid bodies (EBs). We report here that Activin B in a dose depend manner markedly up-regulates Pdx1 expression as compared to Activin A and untreated cultures. Pdx1(+) cells co-express FOXA2 but lacks, however, co-expression with nkx6.1, a marker combination that in the present study is shown precisely to identify embryonic and fetal pancreas anlage in humans. Pdx1(+) cells are found in cell clusters also expressing Serpina1 and FABP1, suggesting activation of intestinal/liver developmental programs. Moreover, Activin B up-regulates Sonic Hedgehog (Shh) and its target Gli1, which during normal development is suppressed in the pancreatic anlage. In conclusion, Activin B is a potent inducer of Pdx1 as well as Shh in differentiating hESCs. The data suggest that additional suppression of Shh signaling may be required to allow for proper specification of pancreatic cell lineages in hESCs.     

BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies

The vasculature develops primarily through two processes, vasculogenesis and angiogenesis. Although much work has been published on angiogenesis, less is known of the mechanisms regulating the de novo formation of the vasculature commonly called vasculogenesis. Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health-registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium-2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P<0.05). Analysis of the expression of 45 genes by quantitative real time-polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (alpha-smooth muscle actin [alpha SMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.     

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

Cancer genes hypermethylated in human embryonic stem cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.     

Three-dimensional porous alginate scaffolds provide a conducive environment for generation of well-vascularized embryoid bodies from human embryonic stem cells

Differentiation of human embryonic stem cells (hESCs) can be instigated through the formation of embryo-like aggregates in suspension, termed human embryoid bodies (hEBs). Controlling cell aggregation and agglomeration during hEBs formation has a profound effect on the extent of cell proliferation and differentiation. In a previous work, we showed that control over hEBs formation and differentiation can be achieved via cultivation of hESC suspensions in a rotating bioreactor system. We now report that hEBs can be generated directly from hESC suspensions within three-dimensional (3D) porous alginate scaffolds. The confining environments of the alginate scaffold pores enabled efficient formation of hEBs with a relatively high degree of cell proliferation and differentiation; encouraged round, small-sized hEBs; and induced vasculogenesis in the forming hEBs to a greater extent than in static or rotating cultures. We therefore conclude that differentiation of hEBs can be induced and directed by physical constraints in addition to chemical cues.     

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.     

Growth and differentiation of embryoid bodies derived from human embryonic stem cells: effect of glucose and basic fibroblast growth factor

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.