Endodermal Cells Research Articles

A major role of class III HD-ZIPs in promoting sugar beet cyst nematode parasitism in Arabidopsis.

Cyst nematodes use a stylet to secrete CLE-like peptide effector mimics into selected root cells of their host plants to hijack endogenous plant CLE signaling pathways for feeding site (syncytium) formation. Here, we identified ATHB8, encoding a HD-ZIP III family transcription factor, as a downstream component of the CLE signaling pathway in syncytium formation. ATHB8 is expressed in the early stages of syncytium initiation, and then transitions to neighboring cells of the syncytium as it expands; an expression pattern coincident with auxin response at the infection site. Conversely, MIR165a, which expresses in endodermal cells and moves into the vasculature to suppress HD-ZIP III TFs, is down-regulated near the infection site. Knocking down HD-ZIP III TFs by inducible over-expression of MIR165a in Arabidopsis dramatically reduced female development of the sugar beet cyst nematode (Heterodera schachtii). HD-ZIP III TFs are known to function downstream of auxin to promote cellular quiescence and define stem cell organizer cells in vascular patterning. Taken together, our results suggest that HD-ZIP III TFs function together with a CLE and auxin signaling network to promote syncytium formation, possibly by inducing root cells into a quiescent status and priming them for initial syncytial cell establishment and/or subsequent cellular incorporation.

A Single Cell Transcriptomic Fingerprint of Stressed Premature, Imbalanced Differentiation of Embryonic Stem Cells.

Miscarriages cause a greater loss-of-life than cardiovascular diseases, but knowledge about environmentally induced miscarriages is limited. Cultured naïve pluripotent embryonic stem cells (ESC) differentiate into extra-embryonic endoderm/extraembryonic endoderm (XEN) or formative pluripotent ESC, during the period emulating maximal miscarriage of peri-implantation development. In previous reports using small marker sets, hyperosmotic sorbitol, or retinoic acid (RA) decreased naïve pluripotency and increased XEN by FACS quantitation. Bulk and single cell (sc)RNAseq analyses of two cultured ESC lines was done, corroborated by qPCR. Transcriptomic responses were analyzed of cultured ESC stressed by Sorbitol, with Leukemia inhibitory factor (LIF + ; stemness growth factor), RA without LIF to control for XEN induction, and compared with normal differentiation (LIF - , ND). Sorbitol and RA increase subpopulations of 2-cell embryo-like (2CEL) and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE) cells and suppress formative pluripotency, imbalancing alternate lineage choices of initial naïve pluripotent cultured ESC compared with ND. Although bulk RNAseq and gene ontology (GO) group analyses suggest that stress induces anterior VE-head organizer and placental markers, scRNAseq reveals relatively few cells. But VE and placental markers/cells were in adjacent stressed cell clusters in the UMAP, like recent, normal UMAP of conceptuses. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance. Hyperosmotic stress, and other toxicological stresses, like drugs with active ingredient RA, may cause premature, lineage imbalance, resulting in miscarriages or birth defects.

Mature mediastinal teratoma with adhesions to the pericardium: A rare case report.

Mature mediastinal teratoma (MMT) is a benign tumor that is composed of well-differentiated tissues from all three germ cell layers. Malignant tumors have a distinct feature of adhering to the surrounding organs. Therefore, adhesion of MMT to the adjacent tissues, as a benign tumor, is rare and considered a surgical challenge. It leads to incomplete tumor resection to avoid damage to the surrounding tissues. MMT is a germ cell tumor that contains well-differentiated tissues from ectodermal, mesodermal, and endodermal germ cell layers. MMT comprises approximately 3%-12% of mediastinal tumors in adults and 60%-70% of mediastinal germ cell tumors. Complete surgical resection is the standard treatment. When adhesion to the surrounding tissues is present, residual tissues could be retained to reduce the injury of the peripheral blood vessels and nerves. We are reporting a rare case of MMT with adhesions to the pericardium and discussing the diagnostic and surgical challenges.

The Drosophila adult midgut progenitor cells arise from asymmetric divisions of neuroblast-like cells.

The Drosophila adult midgut progenitor cells (AMPs) give rise to all cells in the adult midgut epithelium, including the intestinal stem cells (ISCs). While they share many characteristics with the ISCs, it remains unclear how they are generated in the early embryo. Here, we show that they arise from a population of endoderm cells, which exhibit multiple similarities with Drosophila neuroblasts. These cells, which we have termed endoblasts, are patterned by homothorax (Hth) and undergo asymmetric divisions using the same molecular machinery as neuroblasts. We also show that the conservation of this molecular machinery extends to the generation of the enteroendocrine lineages. Parallels have previously been drawn between the pupal ISCs and larval neuroblasts. Our results suggest that these commonalities exist from the earliest stages of specification of progenitor cells of the intestinal and nervous systems and may represent an ancestral pathway for multipotent progenitor cell specification.

Synthetic hydrogel substrate for human induced pluripotent stem cell definitive endoderm differentiation

Human induced pluripotent stem cells (hiPSCs) can give rise to multiple lineages derived from three germ layers, endoderm, mesoderm and ectoderm. Definitive endoderm (DE) cell types and tissues have great potential for regenerative medicine applications. Current hiPSC differentiation protocols focus on the addition of soluble factors; however, extracellular matrix properties are known to also play a role in dictating cell fate. Matrigel™ is the gold standard for DE differentiation, but this xenogeneic, poorly defined basement membrane extract limits the clinical translatability of DE-derived tissues. Here we present a fully defined PEG-based hydrogel substrate to support hiPSC-derived DE differentiation. We screened hydrogel formulations presenting different adhesive peptides and matrix stiffness. Our results demonstrate that presenting a short peptide, cyclic RGD, on the engineered PEG hydrogel supports the transition from undifferentiated hiPSCs to DE using a serum-free, commercially available kit. We show that increasing substrate stiffness (G’ = 1.0–4.0 kPa) results in an increased linear response in DE differentiation efficiency. We also include a temporal analysis of the expression of integrin and syndecan receptors as the hiPSCs undergo specification towards DE lineage. Finally, we show that focal adhesion kinase activity regulates hiPSC growth and DE differentiation efficiency. Overall, we present a fully defined matrix as a synthetic alternative for Matrigel™ supporting DE differentiation.

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells.

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.

Inhibition of proteasome activity facilitates definitive endodermal specification of pluripotent stem cells by influencing YAP signalling

AimsThe knowledge of the molecular players that regulate the generation of endoderm cells is imperative to obtain homogenous population of pancreatic β-cells from stem cells. The Ubiquitin proteasome system (UPS) has been envisaged as a crucial intracellular protein degradation system, but its role in the generation of β-cells remains elusive. Hence, it would be appropriate to unravel the potential role of UPS in endoderm specification and utilize the understanding to generate β-cells from pluripotent stem cells. Materials and methodsThe pluripotent stem cells (mESCs, miPSCs and hIPSCs) were subjected to differentiation towards pancreatic β-cells and assessed the proteasomal activity during endodermal differentiation. Pharmacologic agents MG132 and IU-1 were employed to inhibit and activate proteasomal activity respectively at the definitive endoderm stage to investigate its impact on the generation of β-cells. The expression of stage-specific genes were analyzed at transcript and protein levels. We also explored the role of unfolded protein response and UPS-regulated signalling pathways in endodermal differentiation. Key findingsWe observed decreased proteasomal activity specifically during endoderm, but not during the generation of other lineages. Extraneous proteasomal inhibition enhanced the expression of endodermal genes while increasing the proteasomal activity hindered definitive endodermal differentiation. Proteasomal inhibition at the definitive endodermal stage culminated in an enriched generation of insulin-positive cells. Elevated endodermal gene expression was consistent in mESCs and hIPSCs upon proteasomal inhibition. Mechanistic insight revealed the proteasome-inhibited enhanced endodermal differentiation to be via modulating the YAP pathway. SignificanceOur study unravels the specific involvement of UPS in endoderm cell generation from pluripotent stem cells and paves the way for obtaining potential definitive endodermal cells for plausible cellular therapy in the future.

Gα13 controls pharyngeal endoderm convergence by regulating E-cadherin expression and RhoA activation.

Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoA-mediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13 mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endoderm C&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis.

Single-cell analysis of preimplantation embryonic development in guinea pigs

BackgroundGuinea pigs exhibit numerous physiological similarities to humans, yet the details of their preimplantation embryonic development remain largely unexplored.ResultsTo address this, we conducted single-cell sequencing on the transcriptomes of cells isolated from the zygote stage through preimplantation stages in guinea pigs. This study identified seven distinct cell types within guinea pig preimplantation embryos and pinpointed the timing of zygotic gene activation (ZGA). Trajectory analysis revealed a bifurcation into two lineage-specific branches, accompanied by alterations in specific pathways, including oxidative phosphorylation and vascular endothelial growth factor (VEGF). Additionally, co-expressed gene network analysis highlighted the most enriched functional modules for the epiblast (EPI), primitive endoderm (PrE), and inner cell mass (ICM). Finally, we compared the similarities and differences between human and guinea pig epiblasts (EPIs).ConclusionThis study systematically constructs a cell atlas of guinea pig preimplantation embryonic development, offering fresh insights into mammalian embryonic development and providing alternative experimental models for studying human embryonic development.

Comparative Analysis of Casparian Strip Membrane Domain Protein Family in Oryza sativa (L.) and Arabidopsis thaliana (L.).

The Casparian strip membrane domain proteins (CASPs) are pivotal for the formation of the Casparian strip (CS) in endodermal cells and play a crucial role in a plant's response to environmental stresses. However, existing research on the CASP gene family in rice and Arabidopsis lacks a comprehensive bioinformatics analysis and necessitates further exploration. In this study, we identified 41 OsCASP and 39 AtCASP genes, which were grouped into six distinct subgroups. Collinearity analysis underscored the pivotal roles of WGD and TD events in driving the evolution of CASPs, with WGDs being the dominant force. On the one hand, the analysis of cis-elements indicated that most OsCASP and AtCASP genes contain MYB binding motifs. On the other hand, RNA-seq revealed that the majority of OsCASP and AtCASP genes are highly expressed in roots, particularly in endodermal cells, where OsCASP_like11/9 and AtCASP_like1/31 demonstrated the most pronounced expression. These results suggest that OsCASP_like11/9 and AtCASP_like1/31 might be candidate genes involved in the formation of the endodermis CS. RT-qPCR results demonstrated that OsCASP_like2/3/13/17/21/30 may be candidate genes for the ion defect process. Collectively, this study offers a theoretical foundation for unraveling the biological functions of CASP genes in rice and Arabidopsis.

Stepwise de novo establishment of inactive X chromosome architecture in early development.

X chromosome inactivation triggers a dramatic reprogramming of transcription and chromosome architecture. However, how the chromatin organization of inactive X chromosome is established de novo in vivo remains elusive. Here, we identified an Xist-separated megadomain structure (X-megadomains) on the inactive X chromosome in mouse extraembryonic lineages and extraembryonic endoderm (XEN) cell lines, and transiently in the embryonic lineages, before Dxz4-delineated megadomain formation at later stages in a strain-specific manner. X-megadomain boundary coincides with strong enhancer activities and cohesin binding in an Xist regulatory region required for proper Xist activation in early embryos. Xist regulatory region disruption or cohesin degradation impaired X-megadomains in extraembryonic endoderm cells and caused ectopic activation of regulatory elements and genes near Xist, indicating that cohesin loading at regulatory elements promotes X-megadomains and confines local gene activities. These data reveal stepwise X chromosome folding and transcriptional regulation to achieve both essential gene activation and global silencing during the early stages of X chromosome inactivation.

Prostatic lineage differentiation from human embryonic stem cells through inducible expression of NKX3-1

BackgroundUnderstanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule.MethodsTo establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses.ResultsWe first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations.ConclusionsThis study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis.

Generation and application of novel hES cell reporter lines for the differentiation and maturation of hPS cell-derived islet-like clusters

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional β-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of β-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as β-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal β-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture.

Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.

Outward askew endodermal cell divisions reveal INFLORESCENCE AND ROOT APICES RECEPTOR KINASE functions in division orientation.

Oriented cell divisions establish plant tissue and organ patterning and produce different cell types; this is particularly true of the highly organized Arabidopsis (Arabidopsis thaliana) root meristem. Mutant alleles of INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) exhibit excess cell divisions in the root endodermis. IRK is a transmembrane receptor kinase that localizes to the outer polar domain of these cells, suggesting that directional signal perception is necessary to repress endodermal cell division. Here, a detailed examination revealed many of the excess endodermal divisions in irk have division planes that specifically skew towards the outer lateral side. Therefore, we termed them 'outward askew' divisions. Expression of an IRK truncation lacking the kinase domain retains polar localization and prevents outward askew divisions in irk; however, the roots exhibit excess periclinal endodermal divisions. Using cell identity markers, we show that the daughters of outward askew divisions transition from endodermal to cortical identity similar to those of periclinal divisions. These results extend the requirement for IRK beyond repression of cell division activity to include cell division plane positioning. Based on its polarity, we propose that IRK at the outer lateral endodermal cell face participates in division plane positioning to ensure normal root ground tissue patterning.

Effects of water stress on apoplastic barrier formation in soil grown roots differ from hydroponically grown roots: Histochemical, biochemical and molecular evidence.

In root research, hydroponic plant cultivation is commonly used and soil experiments are rare. We investigated the response of 12-day-old barley roots, cultivated in soil-filled rhizotrons, to different soil water potentials (SWP) comparing a modern cultivar (cv. Scarlett) with a wild accession ICB181243 from Pakistan. Water potentials were quantified in soils with different relative water contents. Root anatomy was studied using histochemistry and microscopy. Suberin and lignin amounts were quantified by analytical chemistry. Transcriptomic changes were observed by RNA-sequencing. Compared with control with decreasing SWP, total root length decreased, the onset of endodermal suberization occurred much closer towards the root tips, amounts of suberin and lignin increased, and corresponding biosynthesis genes were upregulated in response to decreasing SWP. We conclude that decreasing water potentials enhanced root suberization and lignification, like osmotic stress experiments in hydroponic cultivation. However, in soil endodermal cell suberization was initiated very close towards the root tip, and root length as well as suberin amounts were about twofold higher compared with hydroponic cultivation.

Construction of multilayered small intestine-like tissue by reproducing interstitial flow

Recent advances have made modeling human small intestines invitro possible, but it remains a challenge to recapitulate fully their structural and functional characteristics. We suspected interstitial flow within the intestine, powered by circulating blood plasma during embryonic organogenesis, to be a vital factor. We aimed to construct an invivo-like multilayered small intestinal tissue by incorporating interstitial flow into the system and, in turn, developed the micro-small intestine system by differentiating definitive endoderm and mesoderm cells from human pluripotent stem cells simultaneously on a microfluidic device capable of replicating interstitial flow. This approach enhanced cell maturation and led to the development of a three-dimensional small intestine-like tissue with villi-like epithelium and an aligned mesenchymal layer. Our micro-small intestine system not only overcomes the limitations of conventional intestine models but also offers a unique opportunity to gain insights into the detailed mechanisms underlying intestinal tissue development.

Light-stabilized GIL1 suppresses PIN3 activity to inhibit hypocotyl gravitropism.

Light and gravity coordinately regulate the directional growth of plants. Arabidopsis Gravitropic in the Light 1 (GIL1) inhibits the negative gravitropism of hypocotyls in red and far-red light, but the underlying molecular mechanisms remain elusive. Our study found that GIL1 is a plasma membrane-localized protein. In endodermal cells of the upper part of hypocotyls, GIL1 controls the negative gravitropism of hypocotyls. GIL1 directly interacts with PIN3 and inhibits the auxin transport activity of PIN3. Mutation of PIN3 suppresses the abnormal gravitropic response of gil1 mutant. The GIL1 protein is unstable in darkness but it is stabilized by red and far-red light. Together, our data suggest that light-stabilized GIL1 inhibits the negative gravitropism of hypocotyls by suppressing the activity of the auxin transporter PIN3, thereby enhancing the emergence of young seedlings from the soil.

Supragel-mediated efficient generation of pancreatic progenitor clusters and functional glucose-responsive islet-like clusters

Although several synthetic hydrogels with defined stiffness have been developed to facilitate the proliferation and maintenance of human pluripotent stem cells (hPSCs), the influence of biochemical cues in lineage-specific differentiation and functional cluster formation has been rarely reported. Here, we present the application of Supragel, a supramolecular hydrogel formed by synthesized biotinylated peptides, for islet-like cluster differentiation. We observed that Supragel, with a peptide concentration of 5 mg/mL promoted spontaneous hPSCs formation into uniform clusters, which is mainly attributable to a supporting stiffness of ∼1.5 kPa as provided by the Supragel matrix. Supragel was also found to interact with the hPSCs and facilitate endodermal and subsequent insulin-secreting cell differentiation, partially through its components: the sequences of RGD and YIGSR that interacts with cell membrane molecules of integrin receptor. Compared to Matrigel and suspension culturing conditions, more efficient differentiation of the hPSCs was also observed at the stages 3 and 4, as well as the final stage toward generation of insulin-secreting cells. This could be explained by 1) suitable average size of the hPSCs clusters cultured on Supragel; 2) appropriate level of cell adhesive sites provided by Supragel during differentiation. It is worth noting that the Supragel culture system was more tolerance in terms of the initial seeding densities and less demanding, since a standard static cell culture condition was sufficient for the entire differentiation process. Our observations demonstrate a positive role of Supragel for hPSCs differentiation into islet-like cells, with additional potential in facilitating germ layer differentiation.