Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.     

BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies.

Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, alpha-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

Combined activin A/LiCl/Noggin treatment improves production of mouse embryonic stem cell-derived definitive endoderm cells

Induction of definitive endoderm (DE) cells is a prerequisite for the whole process of embryonic stem (ES) cells differentiating into hepatic or pancreatic progenitor cells. We have established an efficient method to induce mouse ES cell-derived DE cells in suspension embryonic body (EB) culture. Similar to previous studies, mouse ES cell-derived DE cells, which were defined as Cxcr4(+) c-Kit(+) , Cxcr4(+) E-cadherin(+) cells or Cxcr4(+) PDGFRa(-) cells, could be induced in the serum-free EBs at Day 4 of induction. The activations of Wnt, Nodal, and FGF signaling pathways in differentiating EBs promoted DE cell differentiation, while activation of BMP4 signaling inhibited the process. In the present study, we found that chemical activation of canonical Wnt signaling pathway by LiCl could synergize with Activin A-mediated Nodal signaling pathway to promote induction of DE cells, and inhibition of Bmp4 signaling by Noggin along with Activin A/LiCl further improved the efficiency of DE cell differentiation. The derived DE cells were proved for their capacities to become hepatic progenitor cells or pancreatic progenitor cells. In conclusion, we significantly improved the efficiency of generating mouse ES cell-derived DE cells by combined Activin A/LiCl/Noggin treatment. Our work will be greatly helpful to generate ES cell-derived hepatic cells and ES cell-derived pancreatic cells for future regenerative medicine.     

A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies

In order to promote the uniform and reproducible differentiation of human embryonic stem cells (HESCs) in response to exogenously added growth factors, we have developed a method (spin embryoid bodies (EBs)) that uses a recombinant protein-based, animal product-free medium in which HESCs are aggregated by centrifugation to form EBs. In this protocol we describe the formulation of this medium, denoted APEL (Albumin Polyvinylalcohol Essential Lipids), and its use in spin EB differentiation of HESCs. We also describe a more economical variant, BPEL (Bovine Serum Albumin (BSA) Polyvinylalchohol Essential Lipids), in which BSA replaces the recombinant human albumin. The integration of a medium that includes only defined and recombinant components with a defined number of cells to initiate EB formation results in a generally applicable, robust platform for growth factor-directed HESC differentiation.     

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.     

Differentiation of mouse embryonic stem cells into endoderm without embryoid body formation

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.     

FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells

The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.     

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     

The fringe molecules induce endocrine differentiation in embryonic endoderm by activating cMyt1/cMyt3

Endocrine differentiation in the early embryonic pancreas is regulated by Notch signaling. Activated Notch signaling maintains pancreatic progenitor cells in an undifferentiated state, whereas suppression of Notch leads to endocrine cell differentiation. Yet it is not known what mechanism is employed to inactivate Notch in a correct number of precursor cells to balance progenitor proliferation and differentiation. We report that an established Notch modifier, Manic Fringe (Mfng), is expressed in the putative endocrine progenitors, but not in exocrine pancreatic tissues, during early islet differentiation. Using chicken embryonic endoderm as an assaying system, we found that ectopic Mfng expression is sufficient to induce endodermal cells to differentiate towards an endocrine fate. This endocrine-inducing activity depends on inactivation of Notch. Furthermore, ectopic Mfng expression induces the expression of basic helix-loop-helix gene, Ngn3, and two zinc finger genes, cMyt1 and cMyt3. These results suggest that Mfng-mediated repression of Notch signaling could serve as a trigger for endocrine islet differentiation.     

Involvement of Ras in extraembryonic endoderm differentiation of embryonic stem cells

Embryonic stem (ES) cells, derived from the inner cell mass of blastocyst can differentiate into multiple cell lineages. In this study, we examined the possible involvement of Ras in ES cell differentiation. We found that Ras was activated upon formation of embryoid bodies (EBs), an initial step in ES cell differentiation. When expressed during EB differentiation, a dominant-negative mutant of Ras suppressed induction of marker genes for extraembryonic endoderm differentiation, including GATA-4, GATA-6, alpha-fetoprotein, and hepatocyte nuclear factor 3beta, while an activated mutant promoted their induction. Expression of a Ras mutant that selectively activates the Raf/MEK/Erk pathway also enhanced induction of extraembryonic endoderm markers, and treatment with a MEK inhibitor resulted in their decreased expression. In addition, Ras stimulated downregulation of Nanog, a suppressor of endoderm differentiation in ES cells. These data suggest that Ras activation during EB differentiation plays a crucial role in initiation of extraembryonic endoderm differentiation.     

Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells

The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self‐organization into germ layers. In this work, we aimed to investigate how different three‐dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut‐off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification. Biotechnol. Bioeng. 2012; 109: 3119–3132. © 2012 Wiley Periodicals, Inc.     

Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells

The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3–4 days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21–28 days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1⁺ clusters are formed. Almost all cells in PDX1⁺ clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7–14 days, then the number of insulin⁺ cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.     

Stepwise differentiation of human embryonic stem cells into early endoderm derivatives and their molecular characterization

Human embryonic stem cells have the potential to differentiate into all human cell types and therefore hold a great therapeutic promise. Differentiation into the embryonic endoderm and its derivatives is of special interest since it can provide a cure for severe widespread clinical conditions such as diabetes and hepatic failure. In this work we established a unique experimental outline that enables the study of early human endoderm development and can help improve and create new differentiation protocols. To this end we started with mesendoderm cells and separated them into early endoderm and mesoderm progenitor cells using CXCR4 and PDGFRA cell surface markers. We molecularly characterized the different lineages, and demonstrated the importance of the TGFβ pathway in definitive endoderm initiation. The endoderm progenitor cells were then purified creating an endodermal differentiation niche that is not affected by other cell populations. We followed the differentiation of these cells at different time points, and demonstrated an up regulation of genes indicative to differentiation into both foregut and hindgut. Surprisingly, upon continued culture, there was significant down regulation of the hepatic gene signature. This down regulation could be rescued with FGF2 treatment demonstrating its importance in hepatic cell maintenance. In conclusion, we suggest that isolating endoderm progenitor cells is crucial for the analysis of their fate, and enables the identification of factors involved in their differentiation and maintenance.     

Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy, including generation of pancreatic β-cells for diabetes research and treatment. In this study, we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3β inhibitor enhanced efficient endodermal differentiation, and then combined treatment with retinoic acid, a bone morphogenic protein inhibitor, and a transforming growth factor-β (TGF-β) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover, several compounds, including forskolin, dexamethasone, and a TGF-β inhibitor, were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds, more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition, all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol.     

Initial Cell Seeding Density Influences Pancreatic Endocrine Development During in vitro Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers, and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells, to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells, we examined the effect of varying initial cell seeding density from 1.3 x 10⁴ cells/cm² to 5.3 x 10⁴ cells/cm² followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1, PTF1a, NGN3, ARX, and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin, glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression, followed by pancreatic endocrine specification marker expression (BRN4, PAX4, ARX, NEUROD1, NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin, glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment, which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols.     

Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies

Human embryonic stem cell (hESC) differentiation in embryoid bodies (EBs) provides a valuable tool to study the interplay of different germ layers and their influence on cell differentiation. The gene expression of the developing EBs has been shown in many studies, but the protein expression and the spatial composition of different germ layers in human EBs have not been systematically studied. The aim of the present work was to study the temporal and spatial organisation of germ layers based on the expression of mesoderm (Brachyury T), endoderm (AFP) and ectoderm (SOX1) markers during the early stages of differentiation in eight hESC lines. Tissue multi-array technology was applied to study the protein expression of a large number of EBs. According to our results, EB formation and the organisation of germ layers occurred in a similar manner in all the lines. During 12 days of differentiation, all the germ layer markers were present, but no obvious distinct trajectories were formed. However, older EBs were highly organised in structure. Pluripotency marker OCT3/4 expression persisted unexpectedly long in the differentiating EBs. Cavity formation was observed in the immunocytological sections, and caspase-3 expression was high, suggesting a role of apoptosis in hESC differentiation and/or EB formation. The expression of Brachyury T was notably low in all the lines, also those with the best cardiac differentiation capacity, while the expression of SOX1 was higher in some lines, suggesting that the neural differentiation propensity may be detectable already in the early stages of EB differentiation.     

Intestinal lineage commitment of embryonic stem cells

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.