Definitive Endoderm Differentiation Research Articles

Reduction of Activin A gives rise to comparable expression of key definitive endoderm and mature beta cell markers.

Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.

A Comparative Study of Endoderm Differentiation Between Activin A and Small Molecules.

Small molecules such as ROCK inhibitors (Fasudil) and inducer of definitive endoderm 1 (IDE1) can promote differentiation of definitive endoderm, but their effects remain controversial. Therefore, we attempted to verify the effect of these small molecules on promoting definitive endoderm differentiation and found that Fasudil or IDE1 alone could not achieve a similar effect as activin A. On the contrary, CHIR99021 could efficiently promote definitive endoderm differentiation. Nearly 43.4% of experimental cells were SRY-box transcription factor 17 (SOX17)-positive under the synergistic effect of IDE1 and CHIR99021, but its ability to differentiate towards definitive endoderm was still insufficient. Transcriptional analysis and comparison of IDE1 and CHIR99021 synergistic groups (IC) and activin A and CHIR99021 synergistic groups (AC) showed significantly down-regulated definitive endoderm markers in the IC group compared with those in the AC group and the differences between the two groups were mainly due to bone morphogenetic proteins (BMP4) and fibroblast growth factor 17 (FGF17). Further single-cell transcriptome analysis revealed lower expression of BMP4 in SOX17-positive populations, while mothers against decapentaplegic homolog (SMAD) protein translation signal and FGF17 in the AC group were higher than that in the IC group. Western blot analysis showed a significant difference in levels of p-SMAD2/3 between AC and IC groups, which suggests that regulating p-SMAD2/3 may provide a reference to improve the differentiation of definitive endoderm.

Fatty acid synthesis and oxidation regulate human endoderm differentiation by mediating SMAD3 nuclear localization via acetylation.

Metabolic remodeling is one of the earliest events that occur during cell differentiation. Here, we define fatty acid metabolism as a key player in definitive endoderm differentiation from human embryonic stem cells. Fatty acid β-oxidation is enhanced while lipogenesis is decreased, and this is due to the phosphorylation of lipogenic enzyme acetyl-CoA carboxylase by AMPK. More importantly, inhibition of fatty acid synthesis by either its inhibitors or AMPK agonist significantly promotes human endoderm differentiation, while blockade of fatty acid oxidation impairs differentiation. Mechanistically, reduced de novo fatty acid synthesis and enhanced fatty acid β-oxidation both contribute to the accumulation of intracellular acetyl-CoA, which guarantees the acetylation of SMAD3 and further causes nuclear localization to promote endoderm differentiation. Thus, our current study identifies a fatty acid synthesis/oxidation shift during early differentiation and presents an instructive role for fatty acid metabolism in regulating human endoderm differentiation.

Effects of low doses of methylmercury (MeHg) exposure on definitive endoderm cell differentiation in human embryonic stem cells.

Fetal development is one of the most sensitive windows to methylmercury (MeHg) toxicity. Laboratory and epidemiological studies have shown a dose-response relationship between fetal MeHg exposure and neuro performance in different life stages from infants to adults. In addition, MeHg exposure has been reported to be associated with disorders in endoderm-derived organs, such as morphological changes in liver cells and pancreatic cell dysfunctions. However, the mechanisms of the effects of MeHg on non-neuronal organs or systems, especially during the early development of endoderm-derived organs, remain unclear. Here we determined the effects of low concentrations of MeHg exposure during the differentiation of definitive endoderm (DE) cells from human embryonic stem cells (hESCs). hESCs were exposed to MeHg (0, 10, 100, and 200nM) that covers the range of Hg concentrations typically found in human maternal blood during DE cell induction. Transcriptomic analysis showed that sub-lethal doses of MeHg exposure could alter global gene expression patterns during hESC to DE cell differentiation, leading to increased expression of endodermal genes/proteins and the over-promotion of endodermal fate, mainly through disrupting calcium homeostasis and generating ROS. Bioinformatic analysis results suggested that MeHg exerts its developmental toxicity mainly by disrupting ribosome biogenesis during early cell lineage differentiation. This disruption could lead to aberrant growth or dysfunctions of the developing endoderm-derived organs, and it may be the underlying mechanism for the observed congenital diseases later in life. Based on the results, we proposed an adverse outcome pathway for the effects of MeHg exposure during human embryonic stem cells to definitive endoderm differentiation.

A novel method for generating induced pluripotent stem cell (iPSC)-derived alveolar organoids: a comparison of their ability depending on iPSC origin

Background: Alveolar organoids may be useful tools in drug discovery for lung diseases, such as chronic obstructive pulmonary disease, and for studying the effects of respiratory viruses, such as severe acute respiratory syndrome coronavirus 2. Induced pluripotent stem cell (iPSC)-derived alveolar organoids offer ethical and cost-effective alternatives to animal testing and primary cell-based methods. In this study, we present generating alveolar organoids from iPSCs and compare the efficiency of generating iPSCs from alveolar type 2 (AT2) and umbilical cord blood (UCB) cells.Methods: The protocol started with a two-dimensional culture and transitioned to a three-dimensional culture using Matrigel after the endoderm stage. Organoid cultivation lasted for at least 40 days, and the characteristics of alveolar organoids were assessed using flow cytometry, real-time polymerase chain reaction, and immunostaining.Results: iPSCs derived from AT2 cells showed a better ability to generate alveolar organoids than those derived from UCB cells. This difference in the ability of AT2 iPSCs and UCB iPSCs to generate alveolar organoids appeared during the definitive endoderm differentiation stage. AT2 iPSCs showed higher expression of the anterior foregut endoderm marker SOX2 and lung progenitor gene expression markers, such as NKX2.1 and CPM, which are associated with the lung progenitor differentiation stage.Conclusion: This protocol successfully generated alveolar organoids from AT2 iPSCs; however, the efficiency of differentiation varied depending on the origin of the iPSCs. This study also found differences in gene expression and developmental potential between iPSCs, which may have contributed to the observed differences in differentiation efficiency.

Dynamic network-guided CRISPRi screen identifies CTCF-loop-constrained nonlinear enhancer gene regulatory activity during cell state transitions.

Comprehensive enhancer discovery is challenging because most enhancers, especially those contributing to complex diseases, have weak effects on gene expression. Our gene regulatory network modeling identified that nonlinear enhancer gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Using human embryonic stem cell definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. We discovered a comprehensive set of enhancers for each of the core endoderm-specifying transcription factors. Many enhancers had strong effects mid-transition but weak effects post-transition, consistent with the nonlinear temporal responses to enhancer perturbation predicted by the modeling. Integrating three-dimensional genomic information, we were able to develop a CTCF-loop-constrained Interaction Activity model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and systematic enhancer discovery in both normal and pathological cell state transitions.

The developmental toxicity of PM2.5 on the early stages of fetal lung with human lung bud tip progenitor organoids

Exposure to air pollution has been proven to be associated with impaired fetal lung development. However, due to the lack of reliable human source models, it is still challenging to deeply understand the human fetal lung development under PM2.5 exposure. Here, we utilized human embryonic stem cell (hESC) line H9 to generate lung bud tip progenitor organoids (LPOs), a process that mimics early stages of fetal lung development including definitive endoderm (DE) formation, anterior foregut endoderm (AFE) differentiation and lung progenitor cell specification, to evaluate potential pulmonary developmental toxicity of PM2.5. We demonstrated that PM2.5 exposure the entire LPOs induction from hESCs significantly affected cellular proliferation of LPOs, and altered the expression of lung progenitor cell markers NKX2.1, SOX2 and SOX9, which are canonically defined subsequently proximal–distal airways specification. To explore the dynamic influences of PM2.5 exposure at different stages of LPOs specification, we also found that PM2.5 exposure significantly affected the expression of several transcriptional factors that are important for the differentiation of DE and AFE. Mechanistically, we suggested PM2.5-induced developmental toxicity to LPOs was partially linked with the Wnt/β-catenin signaling pathway. Therefore, our findings further emphasize the substantial health risks in the development of respiratory system associated with prenatal exposure to PM2.5.

A desert lncRNA HIDEN regulates human endoderm differentiation via interacting with IMP1 and stabilizing FZD5 mRNA

BackgroundExtensive studies have revealed the function and mechanism of lncRNAs in development and differentiation, but the majority have focused on those lncRNAs adjacent to protein-coding genes. In contrast, lncRNAs located in gene deserts are rarely explored. Here, we utilize multiple differentiation systems to dissect the role of a desert lncRNA, HIDEN (human IMP1-associated "desert" definitive endoderm lncRNA), in definitive endoderm differentiation from human pluripotent stem cells.ResultsWe show that desert lncRNAs are highly expressed with cell-stage-specific patterns and conserved subcellular localization during stem cell differentiation. We then focus on the desert lncRNA HIDEN which is upregulated and plays a vital role during human endoderm differentiation. We find depletion of HIDEN by either shRNA or promoter deletion significantly impairs human endoderm differentiation. HIDEN functionally interacts with RNA-binding protein IMP1 (IGF2BP1), which is also required for endoderm differentiation. Loss of HIDEN or IMP1 results in reduced WNT activity, and WNT agonist rescues endoderm differentiation deficiency caused by the depletion of HIDEN or IMP1. Moreover, HIDEN depletion reduces the interaction between IMP1 protein and FZD5 mRNA and causes the destabilization of FZD5 mRNA, which is a WNT receptor and necessary for definitive endoderm differentiation.ConclusionsThese data suggest that desert lncRNA HIDEN facilitates the interaction between IMP1 and FZD5 mRNA, stabilizing FZD5 mRNA which activates WNT signaling and promotes human definitive endoderm differentiation.

Integrative network analysis of circular RNAs reveals regulatory mechanisms for hepatic specification of human iPSC-derived endoderm

BackgroundHuman-induced pluripotent stem cell (hiPSC)-derived functional hepatic endoderm (HE) is supposed to be an alternative option for replacement therapy for end-stage liver disease. However, the high heterogeneity of HE cell populations is still challenging. Hepatic specification of definitive endoderm (DE) is an essential stage for HE induction in vitro. Recent studies have suggested that circular RNAs (circRNAs) determine the fate of stem cells by acting as competing endogenous RNAs (ceRNAs). To date, the relationships between endogenous circRNAs and hepatic specification remain elusive.MethodsThe identities of DE and HE derived from hiPSCs were determined by qPCR, cell immunofluorescence, and ELISA. Differentially expressed circRNAs (DEcircRNAs) were analysed using the Arraystar Human circRNA Array. qPCR was performed to validate the candidate DEcircRNAs. Intersecting differentially expressed genes (DEGs) of the GSE128060 and GSE66282 data sets and the DEcircRNA-predicted mRNAs were imported into Cytoscape for ceRNA networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were involved in the enrichment analysis. Hepatic markers and Wnt/β-catenin were detected in hsa_circ_004658-overexpressing cells by western blotting. Dual-luciferase reporter assays were used to evaluate the direct binding among hsa_circ_004658, miRNA-1200 and CDX2. DE cells were transfected with miR-1200 mimics, adenovirus containing CDX2, and Wnt/β-catenin was detected by western blotting.ResultshiPSC-derived DE and HE were obtained at 4 and 9 days after differentiation, as determined by hepatic markers. During hepatic specification, 626 upregulated and 208 downregulated DEcircRNAs were identified. Nine candidate DEcircRNAs were validated by qPCR. In the ceRNA networks, 111 circRNA–miRNA–mRNA pairs were involved, including 90 pairs associated with hsa_circ_004658. In addition, 53 DEGs were identified among the intersecting mRNAs of the GSE128060 and GSE66282 data sets and the hsa_circ_004658-targeted mRNAs. KEGG and GO analyses showed that the DEGs associated with hsa_circ_004658 were mainly enriched in the WNT signalling pathway. Furthermore, hsa_circ_004658 was preliminarily verified to promote hepatic specification, as determined by hepatic markers (AFP, ALB, HNF4A, and CK19) (p < 0.05). This promotive effect may be related to the inhibition of the Wnt/β-catenin signalling pathway (detected by β-catenin, p-β-catenin, and TCF4) when hsa_circ_004658 was overexpressed (p < 0.05). Dual-luciferase reporter assays showed that there were binding sites for miR-1200 in the hsa_circ_004658 sequence, and confirmed the candidate DEG (CDX2) as a miR-1200 target. The level of miR-1200 decreased and the level of CDX2 protein expression increased when hsa_circ_004658 was overexpressed (p < 0.05). In addition, the results showed that CDX2 may suppress the Wnt/β-catenin signalling during hepatic specification (p < 0.05).ConclusionsThis study analysed the profiles of circRNAs during hepatic specification. We identified the hsa_circ_004658/miR-1200/CDX2 axis and preliminarily verified its effect on the Wnt/β-catenin signalling pathway during hepatic specification. These results provide novel insight into the molecular mechanisms involved in hepatic specification and could improve liver development in the future.Graphical

SOX transcription factors direct TCF-independent WNT/β-catenin responsive transcription to govern cell fate in human pluripotent stem cells.

WNT/β-catenin signaling controls gene expression across biological contexts from development and stem cell homeostasis to diseases including cancer. How β-catenin is recruited to distinct enhancers to activate context-specific transcription is unclear, given that most WNT/ß-catenin-responsive transcription is thought to be mediated by TCF/LEF transcription factors (TFs). With time-resolved multi-omic analyses, we show that SOX TFs can direct lineage-specific WNT-responsive transcription during the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm and neuromesodermal progenitors. We demonstrate that SOX17 and SOX2 are required to recruit β-catenin to lineage-specific WNT-responsive enhancers, many of which are not occupied by TCFs. At TCF-independent enhancers, SOX TFs establish a permissive chromatin landscape and recruit a WNT-enhanceosome complex to activate SOX/ß-catenin-dependent transcription. Given that SOX TFs and the WNT pathway are critical for specification of most cell types, these results have broad mechanistic implications for the specificity of WNT responses across developmental and disease contexts.

Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

SummaryA major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

Generation of Urothelial Cells from Mouse-Induced Pluripotent Stem Cells.

The search for a suitable alternative for urethral defect is a challenge in the field of urethral tissue engineering. Induced pluripotent stem cells (iPSCs) possess multipotential for differentiation. The in vitro derivation of urothelial cells from mouse-iPSCs (miPSCs) has thus far not been reported. The purpose of this study was to establish an efficient and robust differentiation protocol for the differentiation of miPSCs into urothelial cells. Our protocol made the visualization of differentiation processes of a 2-step approach possible. We firstly induced miPSCs into posterior definitive endoderm (DE) with glycogen synthase kinase-3β (GSK3β) inhibitor and Activin A. We investigated the optimal conditions for DE differentiation with GSK3β inhibitor treatment by varying the treatment time and concentration. Differentiation into urothelial cells, was directed with all-trans retinoic acid (ATRA) and recombinant mouse fibroblast growth factor-10 (FGF-10). Specific markers expressed at each stage of differentiation were validated by flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR) assay, immunofluorescence staining, and western blotting Assay. The miPSC-derived urothelial cells were successfully in expressed urothelial cell marker genes, proteins, and normal microscopic architecture. We built a model of directed differentiation of miPSCs into urothelial cells, which may provide the evidence for a regenerative potential of miPSCs in preclinical animal studies.

Regeneration of insulin-producing cells from iPS cells using functionalized scaffolds and solid lipid nanoparticles

BackgroundFrom ongoing human clinical trials, cell therapy for diabetic treatment using islet-like organoid generation from induced pluripotent stem cells (iPSCs) appears to have a promising future. MethodsDevelopment of islet cells from iPSCs was guided in activin A- and Wnt3a-grafted gelatin-polyacrylamide inverted colloidal crystal scaffolds (activin A-Wnt3a-Gel-PAAM ICCS) with pore sizes of 70 and 160 µm, followed by regulation with fibroblast growth factor 7-grafted retinoic acid-entrapped solid lipid nanoparticles (FGF7-RA-SLNs). Significant FindingsFGF7-RA-SLNs diffused into activin A-Wnt3a-Gel-PAAM ICCS, and controlled differentiation of definitive endoderm (DE) cells into islet cells. The immunochemical staining, flow cytometry and western blot demonstrated combined effects among activin A and Wnt3a, FGF7 and RA in materials for the 2-step differentiation. Wnt3a on the pore surface promoted activin A activity for generating DE cells, and FGF7 enhanced RA activity for producing islet cells. Under glucose stimulation, activin A-Wnt3a-Gel-PAAM ICCS with pore sizes of 70 µm were prone to regenerate insulin-producing β-cells rather than α cells. The optimized conditions for insulin secretion from β-like cells in this study were pore size of 70 µm, Gel:PAAM of 7:3, 100 ng/mL activin A, 100 ng/mL Wnt3a, 50 ng/mL FGF7 and 600 ng/mL RA. Activin A-Wnt3a-Gel-PAAM ICCS associated with FGF7-RA-SLNs can be effective in forming biomimetic pancreas and supporting cell therapy from iPSCs for diabetic management.

Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells

Cellular organelles play fundamental roles in almost all cell behaviors. Mitochondria have been reported to be functionally linked to various biological processes, including reprogramming and pluripotency maintenance. However, very little about the role of mitochondria has been revealed in human early development and lineage specification. Here, we reported the characteristics and function of mitochondria during human definitive endoderm differentiation. Using a well-established differentiation system, we first investigated the change of mitochondrial morphology by comparing undifferentiated pluripotent stem cells, the intermediate mesendoderm cells, and differentiated endoderm cells, and found that mitochondria were gradually elongated and matured along differentiation. We further analyzed the expression pattern of mitochondria-related genes by RNA-seq, indicating that mitochondria became active during differentiation. Supporting this notion, the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS) was increased as well. Functionally, we utilized chemicals and genome editing techniques, which could interfere with mitochondrial homeostasis, to determine the role of mitochondria in human endoderm differentiation. Treatment with mitochondrial inhibitors, or genetic depletion of mitochondrial transcription factor A (TFAM), significantly reduced the differentiation efficiency of definitive endoderm. In addition, the defect in endoderm differentiation due to dysfunctional mitochondria could be restored to some extent by the addition of ATP. Moreover, the clearance of excessive ROS due to dysfunctional mitochondria by N-acetylcysteine (NAC) improved the differentiation as well. We further found that ATP and NAC could partially replace the growth factor activin A for definitive endoderm differentiation. Our study illustrates the essential role of mitochondria during human endoderm differentiation through providing ATP and regulating ROS levels, which may provide new insight for metabolic regulation of cell fate determination.

Parallel functional assessment of m6A sites in human endodermal differentiation with base editor screens

N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.

Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

The small intestinal epithelium has an important role in nutrition, but also in drug absorption and metabolism. There are a few two-dimensional (2D) patient-derived induced pluripotent stem cell (iPSC)-based intestinal models enabling easy evaluation of transcellular transport. It is known that animal-derived components induce variation in the experimental outcomes. Therefore, we aimed to refine the differentiation protocol by using animal-free components. More specifically, we compared maturation of 2D-cultured iPCSs toward small intestinal epithelial cells when cultured either with or without serum, and either on Geltrex or on animal-free, recombinant laminin-based substrata. Differentiation status was characterized by qPCR, immunofluorescence imaging, and functionality assays. Our data suggest that differentiation toward definitive endoderm is more efficient without serum. Both collagen- and recombinant laminin-based coating supported differentiation of definitive endoderm, posterior definitive endoderm, and small intestinal epithelial cells from iPS-cells equally well. Small intestinal epithelial cells differentiated on recombinant laminin exhibited slightly more enterocyte specific cellular functionality than cells differentiated on Geltrex. Our data suggest that functional small intestinal epithelial cells can be generated from iPSCs in serum-free method on xeno-free substrata. This method is easily converted to an entirely xeno-free method.

Emergence and patterning dynamics of mouse-definitive endoderm

SummaryThe segregation of definitive endoderm (DE) from bipotent mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE commitment and onset has been challenging as it occurs within a narrow spatiotemporal window in the embryo. Here, we employ a dual Bra/Sox17 reporter cell line to study DE onset dynamics. We find Sox17 expression initiates in vivo in isolated cells within a temporally restricted window. In 2D and 3D in vitro models, DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17 + cells. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Self-sorting coincides with upregulation of E-cadherin but is not necessary for DE differentiation or proliferation. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.

MiR-27b antagonizes BMP signaling in early differentiation of human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.

Generation of pancreatic progenitors from human pluripotent stem cells by small molecules.

SummaryHuman pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) provide promising cell therapies for type 1 diabetes. Current PP differentiation requires a high amount of Activin A during the definitive endoderm (DE) stage, making it economically difficult for commercial ventures. Here we identify a dose-dependent role for Wnt signaling in controlling DE differentiation without Activin A. While high-level Wnt activation induces mesodermal formation, low-level Wnt activation by a small-molecule inhibitor of glycogen synthase kinase 3 is sufficient for DE differentiation, yielding SOX17+FOXA2+ DE cells. BMP inhibition further enhances this DE differentiation, generating over 87% DE cells. These DE cells could be further differentiated into PPs and functional β cells. RNA-sequencing analysis of PP differentiation from hPSCs revealed expected transcriptome dynamics and new gene regulators during our small-molecule PP differentiation protocol. Overall, we established a robust growth-factor-free protocol for generating DE and PP cells, facilitating scalable production of pancreatic cells for regenerative applications.

Emergence and Patterning Dynamics of Mouse Definitive Endoderm

The segregation of definitive endoderm (DE) from bi-potent mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE commitment and onset has been challenging as it occurs within a narrow spatio-temporal window in the embryo. Here we employ a novel dual Bra/Sox17 reporter cell line to study DE onset dynamics. We find Sox17 expression initiates in a few isolated cells in vivo within a temporally restricted window. Using 2D and 3D in vitro models, we show that DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17+ cells. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Self-sorting coincides with up-regulation of E-cadherin but is not necessary for DE differentiation or proliferation. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.