Ectodermal Lineages Research Articles

UBR-5 and UBE2D mediate timely exit from stem fate via destabilization of poly(A)-binding protein PABP-2 in cell state transition

UBR5 E3 ligase has been associated with cancer susceptibility and neuronal integrity, with functions in chromatin regulation and proteostasis. However, the functions of ubr5 within animals remain unclear due to lethality in both mammals and flies when disrupted. Using Caenorhabditis elegans, we show that UBR-5 E3 ligase is required for timely exit of stem fate and complete transition into multiple cell type descendants in an ectodermal blast lineage. Animals lacking intact UBR-5 function simultaneously exhibit both stem fate and differentiated fate in the same descendant cells. A functional screen of UBR-5 physical interactors allowed us to identify the UBE2D2/3 E2 conjugase LET-70 working with UBR-5 to exit stem fate. Strikingly, we revealed that another UBR-5 physical interactor, namely the nuclear poly(A)-binding protein PABPN1 ortholog PABP-2, worked antagonistically to UBR-5 and LET-70. Lowering pabp-2 levels restored normal transition of cell state out of stemness and promoted normal cell fusion when either ubr-5 or let-70 UBE2D function was compromised. The UBR-5-LET-70 and PABP-2 switch works independently of the stem pool size determined by pluripotency factors like lin-28. UBR-5 limits PABP-2 protein and reverses the PABP-2-dependent gene expression program including developmental, proteostasis, and innate immunity genes. Loss of ubr-5 rescues the developmental stall when pabp-2 is compromised. Disruption of ubr-5 elevates PABP-2 levels and prolongs expression of ectodermal and muscle stem markers at the transition to adulthood. Additionally, ubr-5 mutants exhibit an extended period of motility during aging and suppress pabp-2-dependent early onset of immobility.

Deciphering the dynamic single-cell transcriptional landscape in the ocular surface ectoderm differentiation system

Abstract The ocular surface ectoderm (OSE) is essential for the development of the ocular surface, yet the molecular mechanisms driving its differentiation are not fully understood. In this study, we used single-cell transcriptomic analysis to explore the dynamic cellular trajectories and regulatory networks during the in vitro differentiation of embryonic stem cells (ESCs) into the OSE lineage. We identified nine distinct cell subpopulations undergoing differentiation along three main developmental branches: neural crest, neuroectodermal, and surface ectodermal lineages. Key marker gene expression, transcription factor activity, and signaling pathway insights revealed stepwise transitions from undifferentiated ESCs to fate-specified cell types, including a PAX6 + TP63 + population indicative of OSE precursors. Comparative analysis with mouse embryonic development confirmed the model’s accuracy in mimicking in vivo epiblast-to-surface ectoderm dynamics. By integrating temporal dynamics of transcription factor activation and cell–cell communication, we constructed a comprehensive molecular atlas of the differentiation pathway from ESCs to distinct ectodermal lineages. This study provides new insights into the cellular heterogeneity and regulatory mechanisms of OSE development, aiding the understanding of ocular surface biology and the design of cell-based therapies for ocular surface disorders.

Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells.

Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.

Early transcriptional similarities between two distinct neural lineages during ascidian embryogenesis

In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are ontogenically associated with ectodermal lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we asked to what extent the transcriptional states of the different neural lineages become similar during the course of progressive lineage restriction. We performed single-cell RNA sequencing (scRNA-seq) analyses on hand-dissected neural precursor cells of the two distinct lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from the 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage, including miR-124, Celf3.a, Zic.r-b, and Ets1/2. Altogether, the current study has revealed genome-wide transcriptional dynamics of neural progenitor cells of two distinct developmental origins. Our scRNA-seq dataset is unique and provides a valuable resource for future analyses, enabling a precise temporal resolution of cell types not previously described from dissociated embryos.

Improved Neural Inductivity of Size-Controlled 3D Human Embryonic Stem Cells Using Magnetic Nanoparticles.

Background: To improve the efficiency of neural development from human embryonic stem cells, human embryoid body (hEB) generation is vital through 3-dimensional formation. However, conventional approaches still have limitations: long-term cultivation and laborious steps for lineage determination. Methods: In this study, we controlled the size of hEBs for ectodermal lineage specification using cell-penetrating magnetic nanoparticles (MNPs), which resulted in reduced time required for initial neural induction. The magnetized cells were applied to concentrated magnetic force for magnet-derived multicellular organization. The uniformly sized hEBs were differentiated in neural induction medium (NIM) and suspended condition. This neurally induced MNP-hEBs were compared with other groups. Results: As a result, the uniformly sized MNP-hEBs in NIM showed significantly improved neural inductivity through morphological analysis and expression of neural markers. Signaling pathways of the accelerated neural induction were detected via expression of representative proteins; Wnt signaling, dopaminergic neuronal pathway, intercellular communications, and mechanotransduction. Consequently, we could shorten the time necessary for early neurogenesis, thereby enhancing the neural induction efficiency. Conclusion: Overall, this study suggests not only the importance of size regulation of hEBs at initial differentiation stage but also the efficacy of MNP-based neural induction method and stimulations for enhanced neural tissue regeneration.

Single-cell RNA sequencing of mid-to-late stage spider embryos: new insights into spider development

BackgroundThe common house spider Parasteatoda tepidariorum represents an emerging new model organism of arthropod evolutionary and developmental (EvoDevo) studies. Recent technical advances have resulted in the first single-cell sequencing (SCS) data on this species allowing deeper insights to be gained into its early development, but mid-to-late stage embryos were not included in these pioneering studies.ResultsTherefore, we performed SCS on mid-to-late stage embryos of Parasteatoda and characterized resulting cell clusters by means of in-silico analysis (comparison of key markers of each cluster with previously published information on these genes). In-silico prediction of the nature of each cluster was then tested/verified by means of additional in-situ hybridization experiments with additional markers of each cluster.ConclusionsOur data show that SCS data reliably group cells with similar genetic fingerprints into more or less distinct clusters, and thus allows identification of developing cell types on a broader level, such as the distinction of ectodermal, mesodermal and endodermal cell lineages, as well as the identification of distinct developing tissues such as subtypes of nervous tissue cells, the developing heart, or the ventral sulcus (VS). In comparison with recent other SCS studies on the same species, our data represent later developmental stages, and thus provide insights into different stages of developing cell types and tissues such as differentiating neurons and the VS that are only present at these later stages.

Role of Hedgehog Signaling Pathways in Multipotent Mesenchymal Stem Cells Differentiation.

Multipotent mesenchymal stem cells (MSCs) have high self-renewal and multi-lineage differentiation potentials and low immunogenicity, so they have attracted much attention in the field of regenerative medicine and have a promising clinical application. MSCs originate from the mesoderm and can differentiate not only into osteoblasts, cartilage, adipocytes, and muscle cells but also into ectodermal and endodermal cell lineages across embryonic layers. To design cell therapy for replacement of damaged tissues, it is essential to understand the signaling pathways, which have a major impact on MSC differentiation, as this will help to integrate the signaling inputs to initiate a specific lineage. Hedgehog (Hh) signaling plays a vital role in the development of various tissues and organs in the embryo. As a morphogen, Hh not only regulates the survival and proliferation of tissue progenitor and stem populations but also is a critical moderator of MSC differentiation, involving tri-lineage and across embryonic layer differentiation of MSCs. This review summarizes the role of Hh signaling pathway in the differentiation of MSCs to mesodermal, endodermal, and ectodermal cells.

SMPD3 expression is spatially regulated in the developing embryo by SOXE factors

During epithelial-to-mesenchymal transition (EMT), significant rearrangements occur in plasma membrane protein and lipid content that are important for membrane function and acquisition of cell motility. To gain insight into how neural crest cells regulate their lipid content at the transcriptional level during EMT, here we identify critical enhancer sequences that regulate the expression of SMPD3, a gene responsible for sphingomyelin hydrolysis to produce ceramide and necessary for neural crest EMT. We uncovered three enhancer regions within the first intron of the SMPD3 locus that drive reporter expression in distinct spatial and temporal domains, together collectively recapitulating the expression domains of endogenous SMPD3 within the ectodermal lineages. We further dissected one enhancer that is specifically active in the migrating neural crest. By mutating putative transcriptional input sites or knocking down upstream regulators, we find that the SOXE-family transcription factors SOX9 and SOX10 regulate the expression of SMPD3 in migrating neural crest cells. Further, ChIP-seq and nascent transcription analysis reveal that SOX10 directly regulates expression of an SMPD3 enhancer specific to migratory neural crest cells. Together these results shed light on how core components of developmental gene regulatory networks interact with metabolic effector genes to control changes in membrane lipid content.

CTCF deletion alters the pluripotency and DNA methylation profile of human iPSCs

Pluripotent stem cells are characterized by their differentiation potential toward endoderm, mesoderm, and ectoderm. However, it is still largely unclear how these cell-fate decisions are mediated by epigenetic mechanisms. In this study, we explored the relevance of CCCTC-binding factor (CTCF), a zinc finger-containing DNA-binding protein, which mediates long-range chromatin organization, for directed cell-fate determination. We generated human induced pluripotent stem cell (iPSC) lines with deletions in the protein-coding region in exon 3 of CTCF, resulting in shorter transcripts and overall reduced protein expression. Chromatin immunoprecipitation showed a considerable loss of CTCF binding to target sites. The CTCF deletions resulted in slower growth and modest global changes in gene expression, with downregulation of a subset of pluripotency-associated genes and neuroectodermal genes. CTCF deletion also evoked DNA methylation changes, which were moderately associated with differential gene expression. Notably, CTCF-deletions lead to upregulation of endo-mesodermal associated marker genes and epigenetic signatures, whereas ectodermal differentiation was defective. These results indicate that CTCF plays an important role in the maintenance of pluripotency and differentiation, especially towards ectodermal lineages.

Assembly of complete mouse embryo models from embryonic and induced stem cell types in vitro.

The interaction between embryonic and extraembryonic tissues is critical in natural mouse embryogenesis. Here, to enable such interaction in vitro, we describe a protocol to assemble a complete mouse embryo model using mouse embryonic stem cells and induced embryonic stem cells to express Cdx2 (or trophoblast stem cells) and Gata4 to reconstitute the epiblast, extraembryonic ectoderm and visceral endoderm lineages, respectively. The resulting complete embryo models recapitulate development from embryonic day 5.0 to 8.5, generating advanced embryonic and extraembryonic tissues that develop through gastrulation to initiate organogenesis to form a head and a beating heart structure as well as a yolk sac and chorion. Once the required stem cell lines are stably maintained in culture, the protocol requires 1 day to assemble complete embryo models and a further 8 days to culture them until headfold stages, although structures can be collected at earlier developmental stages as required. This protocol can be easily performed by researchers with experience in mouse stem cell culture, although they will benefit from knowledge of natural mouse embryos at early postimplantation stages.

Self-assembly of gelatin and collagen in the polyvinyl alcohol substrate and its influence on cell adhesion, proliferation, shape, spreading and differentiation.

Culture substrates display profound influence on biological and developmental characteristic of cells cultured in vitro. This study investigates the influence of polyvinyl alcohol (PVA) substrates blended with different concentration of collagen or/and gelatin on the cell adhesion, proliferation, shape, spreading, and differentiation of stem cells. The collagen/gelatin blended PVA substrates were prepared by air drying. During drying, blended collagen or/and gelatin can self-assemble into macro-scale nucleated particles or branched fibrils in the PVA substrates that can be observed under the optical microscope. These collagen/gelatin blended substrates revealed different surface topography, z-average, roughness, surface adhesion and Young's modulus as examined by the atomic force microscope (AFM). The results of Fourier transform infrared spectroscopy (FTIR) analysis indicated that the absorption of amide I (1,600-1,700cm-1) and amide II (1,500-1,600cm-1) groups increased with increasing collagen and gelatin concentration blended and the potential of fibril formation. These collagen or/and gelatin blended PVA substrates showed enhanced NIH-3T3 fibroblast adhesion as comparing with the pure PVA, control tissue culture polystyrene, conventional collagen-coated and gelatin-coated wells. These highly adhesive PVA substrates also exhibit inhibited cell spreading and proliferation. It is also found that the shape of NIH-3T3 fibroblasts can be switched between oval, spindle and flattened shapes depending on the concentration of collagen or/and gelatin blended. For inductive differentiation of stem cells, it is found that number and ration of neural differentiation of rat cerebral cortical neural stem cells increase with the decreasing collagen concentration in the collagen-blended PVA substrates. Moreover, the PVA substrates blended with collagen or collagen and gelatin can efficiently support and conduct human pluripotent stem cells to differentiate into Oil-Red-O- and UCP-1-positive brown-adipocyte-like cells via ectodermal lineage without the addition of mitogenic factors. These results provide a useful and alternative platform for controlling cell behavior in vitro and may be helpful for future application in the field of regenerative medicine and tissue engineering.

Neural fate commitment of rat full-term amniotic fluid stem cells via three-dimensional embryoid bodies and neurospheres formation.

Full-term amniotic fluid stem cell (AFSC) is an underexplored reserve of broadly multipotent stem cells with potential applications in cell replacement therapy. One aspect worth exploring is the potential of AFSCs to differentiate into neural lineages. Previously, we have shown that full-term AFSC lines established from term gestation amniotic fluid, known as R3 and R2, differentiated into neural lineage through the monolayer adherent method suggesting their neurogenic potential. The neural commitment of the cells through the formation of multicellular aggregates has never been shown before. Here, we explored the ability of R3 to commit to neural fate via the formation of three-dimensional multicellular aggregates, namely embryoid bodies (EBs) and neurospheres, exhibiting distinct characteristics resembling EBs and neurospheres as obtained from other published pluripotent and neural stem cells (NSCs), respectively. Different cell seeding densities of the cells cultured in their respective induction medium generated two distinct types of aggregates with the appropriate sizes for EBs (300-350µm) and neurospheres (50-100µm). The neurospheres expressed a significantly high level of Nestin than EBs. However, EBs stained positive for TUJ1, suggesting the presence of early post-mitotic neurons representing the ectodermal lineage. In contrast, the presence of the NSC population in neurosphere culture was validated with positive expression of Sox1. Notably, dissociated cells from both aggregates differentiated into MAP2-positive neural cells, highlighting the ability of both types of multicellular aggregates to commit to the neural fate. In conclusion, this study highlights the first evidence of neurosphere formation from full-term AFSCs in addition to neural fate commitment via EBs formation. Findings from this study allow researchers to select the suitable approach for neural cell generation and expansion according to research needs.

Early Differentiation Signatures in Human Induced Pluripotent Stem Cells Determined by Non-Targeted Metabolomics Analysis

Human induced pluripotent stem cells (hiPSCs) possess immense potential as a valuable source for the generation of a wide variety of human cells, yet monitoring the early cell differentiation towards a specific lineage remains challenging. In this study, we employed a non-targeted metabolomic analysis technique to analyze the extracellular metabolites present in samples as small as one microliter. The hiPSCs were subjected to differentiation by initiating culture under the basal medium E6 in combination with chemical inhibitors that have been previously reported to direct differentiation towards the ectodermal lineage such as Wnt/β-catenin and TGF-β kinase/activin receptor, alone or in combination with bFGF, and the inhibition of glycogen kinase 3 (GSK-3), which is commonly used for the diversion of hiPSCs towards mesodermal lineage. At 0 h and 48 h, 117 metabolites were identified, including biologically relevant metabolites such as lactic acid, pyruvic acid, and amino acids. By determining the expression of the pluripotency marker OCT3/4, we were able to correlate the differentiation status of cells with the shifted metabolites. The group of cells undergoing ectodermal differentiation showed a greater reduction in OCT3/4 expression. Moreover, metabolites such as pyruvic acid and kynurenine showed dramatic change under ectodermal differentiation conditions where pyruvic acid consumption increased 1–2-fold, while kynurenine secretion decreased 2-fold. Further metabolite analysis uncovered a group of metabolites specifically associated with ectodermal lineage, highlighting the potential of our findings to determine the characteristics of hiPSCs during cell differentiation, particularly under ectodermal lineage conditions.

Establishing extended pluripotent stem cells from human urine cells

BackgroundExtended pluripotent stem cells (EPSCs) can contribute to both embryonic and trophectoderm-derived extraembryonic tissues. Therefore, EPSCs have great application significance for both research and industry. However, generating EPSCs from human somatic cells remains inefficient and cumbersome.ResultsIn this study, we established a novel and robust EPSCs culture medium OCM175 with defined and optimized ingredients. Our OCM175 medium contains optimized concentration of L-selenium-methylcysteine as a source of selenium and ROCK inhibitors to maintain the single cell passaging ability of pluripotent stem cells. We also used Matrigel or the combination of laminin 511 and laminin 521(1:1) to bypass the requirement of feeder cells. With OCM175 medium, we successfully converted integration-free iPSCs from easily available human Urine-Derived Cells (hUC-iPSCs) into EPSCs (O-IPSCs). We showed that our O-IPSCs have the ability to form both intra- and extra- embryonic chimerism, and could contribute to the trophoblast ectoderm lineage and three germ layer cell lineages.ConclusionsIn conclusion, our novel OCM175 culture medium has defined, optimized ingredients, which enables efficient generation of EPSCs in a feeder free manner. With the robust chimeric and differentiation potential, we believe that this system provides a solid basis to improve the application of EPSCs in regenerative medicine.

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation.

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding.

Stiffness-Tunable Hydrogel-Sandwich Culture Modulates the YAP-Mediated Mechanoresponse in Induced-Pluripotent Stem Cell Embryoid Bodies and Augments Cardiomyocyte Differentiation.

Microenvironmental factors, including substrate stiffness, regulate stem cell behavior and differentiation. However, the effects of substrate stiffness on the behavior of induced pluripotent stem cell- (iPSC) derived embryoid bodies (EB) remain unclear. To investigate the effects of mechanical cues on iPSC-EB differentiation, we developed a three-dimensional hydrogel-sandwich culture (HGSC) system that controls the microenvironment surrounding iPSC-EBs using a stiffness-tunable polyacrylamide hydrogel assembly. Mouse iPSC-EBs were seeded between upper and lower polyacrylamide hydrogels of differing stiffness (Young's modulus [E'] = 54.3 ± 7.1kPa [hard], 28.1 ± 2.3kPa [moderate], and 5.1 ± 0.1kPa [soft]) and cultured for 2 days. HGSC induced stiffness-dependent activation of the yes-associated protein (YAP) mechanotransducer and actin cytoskeleton rearrangement in the iPSC-EBs. Moreover, moderate-stiffness HGSC specifically upregulated the mRNA and protein expression of ectoderm and mesoderm lineage differentiation markers in iPSC-EBs via YAP-mediated mechanotransduction. Pretreatment of mouse iPSC-EBs with moderate-stiffness HGSC promoted cardiomyocyte differentiation and structural maturation of myofibrils. Our proposed HGSC system provides a viable platform for investigating the role of mechanical cues on the pluripotency and differentiation of iPSCs that could be beneficial for research into tissue regeneration and engineering. This article is protected by copyright. All rights reserved.

GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development

Ectodermal dysplasias including skin abnormalities and cleft lip/palate result from improper surface ectoderm (SE) patterning. However, the connection between SE gene regulatory networks and disease remains poorly understood. Here, we dissect human SE differentiation with multiomics and establish GRHL2 as a key mediator of early SE commitment, which acts by skewing cell fate away from the neural lineage. GRHL2 and master SE regulator AP2a balance early cell fate output, with GRHL2 facilitating AP2a binding to SE loci. In turn, AP2a restricts GRHL2 DNA binding away from de novo chromatin contacts. Integration of these regulatory sites with ectodermal dysplasia-associated genomic variants annotated within the Biomedical Data Commons identifies 55 loci previously implicated in craniofacial disorders. These include ABCA4/ARHGAP29 and NOG regulatory regions where disease-linked variants directly affect GRHL2/AP2a binding and gene transcription. These studies elucidate the logic underlying SE commitment and deepen our understanding of human oligogenic disease pathogenesis.

Glycolytic Pfkp acts as a Lin41 protein kinase to promote endodermal differentiation of embryonic stem cells.

Unveiling the principles governing embryonic stem cell (ESC) differentiation into specific lineages is critical for understanding embryonic development and for stem cell applications in regenerative medicine. Here, we establish an intersection between LIF-Stat3 signaling that is essential for maintaining murine (m) ESCs pluripotency, and the glycolytic enzyme, the platelet isoform of phosphofructokinase (Pfkp). In the pluripotent state, Stat3 transcriptionally suppresses Pfkp in mESCs while manipulating the cells to lift this repression results in differentiation towards the ectodermal lineage. Pfkp exhibits substrate specificity changes to act as a protein kinase, catalyzing serine phosphorylation of the developmental regulator Lin41. Such phosphorylation stabilizes Lin41 by impeding its autoubiquitination and proteasomal degradation, permitting Lin41-mediated binding and destabilization of mRNAs encoding ectodermal specification markers to favor the expression of endodermal specification genes. This provides new insights into the wiring of pluripotency-differentiation circuitry where Pfkp plays a role in germ layer specification during mESC differentiation.

Development of lacrimal gland organoids from iPSC derived multizonal ocular cells.

Lacrimal gland plays a vital role in maintaining the health and function of the ocular surface. Dysfunction of the gland leads to disruption of ocular surface homeostasis and can lead to severe outcomes. Approaches evolving through regenerative medicine have recently gained importance to restore the function of the gland. Using human induced pluripotent stem cells (iPSCs), we generated functional in vitro lacrimal gland organoids by adopting the multi zonal ocular differentiation approach. We differentiated human iPSCs and confirmed commitment to neuro ectodermal lineage. Then we identified emergence of mesenchymal and epithelial lacrimal gland progenitor cells by the third week of differentiation. Differentiated progenitors underwent branching morphogenesis in the following weeks, typical of lacrimal gland development. We were able to confirm the presence of lacrimal gland specific acinar, ductal, and myoepithelial cells and structures during weeks 4-7. Further on, we demonstrated the role of miR-205 in regulation of the lacrimal gland organoid development by monitoring miR-205 and FGF10 mRNA levels throughout the differentiation process. In addition, we assessed the functionality of the organoids using the β-Hexosaminidase assay, confirming the secretory function of lacrimal organoids. Finally, metabolomics analysis revealed a shift from amino acid metabolism to lipid metabolism in differentiated organoids. These functional, tear proteins secreting human lacrimal gland organoids harbor a great potential for the improvement of existing treatment options of lacrimal gland dysfunction and can serve as a platform to study human lacrimal gland development and morphogenesis.

HCINAP regulates the differentiation of embryonic stem cells by regulating NEDD4 liquid-liquid phase-separation-mediated YAP1 activation

YAP1 functions in lineage differentiation of pluripotent embryonic stem cells (ESCs); however, the detailed mechanisms underlying the regulation of YAP1 activity during ESC differentiation remain elusive. Here, we report that hCINAP serves as a negative regulator of YAP1 during ESC fate decisions. The expression of mCINAP, the murine homolog of hCINAP, is downregulated during the differentiation process of murine ESC (mESC) ectoderm lineage, leading to liquid-liquid phase separation (LLPS) of NEDD4 and activation of YAP1. Mechanistically, hCINAP interacts with and prevents NEDD4 from forming cytoplasmic condensates that compartmentalize YAP1 and its kinase NLK, facilitating YAP1 phosphorylation at Ser128 and promoting YAP1 activation. mCINAP depletion leads to the formation of NEDD4 condensates and YAP1 activation, which impedes endoderm differentiation of mESCs. Our study shows that hCINAP is a vital regulator of YAP1 activity and is essential for stem cell fate decisions, which provides mechanistic insight into early embryogenesis.