Endodermal Lineages Research Articles

Mitochondrial morphology and energy metabolism in reprogrammed porcine expanded potential stem cells.

Expanded potential stem cells (EPSCs) are stem cells that can differentiate into embryonic and extraembryonic lineages, including extraembryonic endoderm and trophoblast lineages. Therefore, EPSCs have great potential in advancing regenerative medicine, elucidating disease mechanisms, and exploring early embryonic development. However, the generation and characterization of EPSCs in pigs have not been thoroughly explored. In this study, we successfully generated porcine EPSCs (pEPSCs). We reprogrammed porcine fetal fibroblasts (PFFs) using an integration-free method with Sendai virus vectors. The resulting pEPSCs expressed key pluripotency markers and demonstrated the ability to differentiate between embryonic and extraembryonic lineages. Notably, reprogramming into pEPSCs was associated with a transformation of mitochondrial morphology from the elongated form observed in PFFs to a globular shape, reflecting potential alterations in energy metabolism. We observed significant remodeling of mitochondrial morphology and a subsequent shift towards glycolytic energy dependence during the reprogramming of PFFs into pEPSCs. Our findings provide valuable insights into the characteristics of EPSCs in pigs and highlight their potential applications in regenerative medicine, disease modeling, and emerging fields such as cell-based meat production.

Depolymerizing F-actin accelerates the exit from pluripotency to enhance stem cell-derived islet differentiation.

Improving generation of insulin-producing islets from human pluripotent stem cells (hPSCs) would enhance their clinical relevance for treating diabetes. Here, we demonstrate that cytoskeletal state at the onset of differentiation is critical for definitive endoderm formation. Depolymerizing F-actin with latrunculin A (latA) during the first 24 hours of differentiation facilitates rapid exit from pluripotency and alters Activin/Nodal, BMP, JNK-JUN, and WNT pathway signaling dynamics during definitive endoderm formation. These signaling changes influence downstream patterning of the gut tube, leading to improved pancreatic progenitor identity and decreased expression of markers for other endodermal lineages. Continued differentiation generates islets containing a higher percentage of β cells that exhibit improved maturation, insulin secretion, and ability to reverse hyperglycemia. Furthermore, this latA treatment reduces enterochromaffin cells in the final cell population and corrects differentiations from hPSC lines that otherwise fail to consistently produce pancreatic islets, highlighting the importance of cytoskeletal signaling during differentiation onset.

PI3K/AKT signaling controls ICM maturation and proper epiblast and primitive endoderm specification in mice.

The inner cell mass (ICM) of early mouse embryos is specified into epiblast (Epi) and primitive endoderm (PrE) lineages during blastocyst formation. The antagonistic transcription factors (TFs) NANOG and GATA-binding protein 6 (GATA6) in combination with fibroblast growth factor (FGF)/extracellular-signal-regulated kinase (ERK) signaling are central actors in ICM fate choice. However, what initiates the specification of ICM progenitors into Epi or PrE and whether other factors are involved in this process has not been fully understood yet. Here, we show that phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) is constitutively active during preimplantation development. Using pharmacological inhibition, we demonstrate that PI3K/AKT enables the formation of a functional ICM capable of giving rise to both the Epi and the PrE: it maintains the expression of the TF NANOG, which specifies the Epi, and confers responsiveness to FGF4, which is essential for PrE specification. Our work thus identifies PI3K/AKT signaling as an upstream regulator controlling the molecular events required for both Epi and PrE specification.

Abstract C078: Chromatin accessibility profiling of human pancreatic tumors reveals epigenetic features of malignant and stromal cell subtypes

Abstract Pancreatic ductal adenocarcinoma (PDAC) disease progression involves complex cell state transitions in both malignant and non-malignant populations within the tumor microenvironment. To uncover PDAC cell states and epigenetic features associated with clinical outcome, we profiled 40 tumor samples (33 primary tumors and 7 metastases) from 39 patients with single cell ATAC-seq, along with 10 sample-matched single cell RNA-seq profiles. Through topic modeling of high dimensional chromatin data, we uncovered distinct cis-regulatory networks underlying the classical and basal-like molecular subtypes of PDAC, which showed differential activity in basal-classical hybrid cells. Furthermore, delineation of epigenetic alterations by basal, classical, and hybrid subtypes revealed a core set of chromatin regions opened in all PDAC subtypes relative to ductal and acinar cells. These universally open regions showed striking overlap with gastrointestinal enhancers, highlighting a common endoderm lineage infidelity process across pancreatic tumors. Within the stromal compartment, we identified novel regulators of myofibroblastic cancer-associated fibroblasts (myCAFs) and inflammatory CAFs (iCAFs) from enhancer networks active in each CAF subtype. Importantly, our data uncover a highly prognostic gene program active in only one of two distinct myCAF chromatin states found in our data, highlighting a putative tumor-promoting myCAF subpopulation. Our work provides a high-resolution description of epigenetic cell state heterogeneity in PDAC, revealing novel regulators and gene programs associated with PDAC subtypes and clinical outcome. Citation Format: Kevin MacPherson-Hawthorne, Jason M. Link, Patrick Worth, Brett Sheppard, Andrew Fields, Colin Daniel, Andrew Nishida, Carl Pelz, Trent Waugh, Ethan Agritelle, John Muschler, Andrew Adey, Rosalie Sears. Chromatin accessibility profiling of human pancreatic tumors reveals epigenetic features of malignant and stromal cell subtypes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C078.

Cell size regulates human endoderm specification through actomyosin-dependent AMOT-YAP signaling

Cell size is a crucial physical property that significantly impacts cellular physiology and function. However, the influence of cell size on stem cell specification remains largely unknown. Here, we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly, cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore, the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements, we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically, the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation, which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation, which is mediated by AMOT nuclear translocation. Additionally, our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation.

Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality.

e-Lysine acetylation is a prominent histone mark found at transcriptionally active loci. Among many lysine acetyl transferases, nonspecific lethal complex (NSL) members are known to mediate the modification of histone H4. In addition to histone modifications, the KAT8 regulatory complex subunit 3 gene (Kansl3), a core member of NSL complex, has been shown to be involved in several other cellular processes such as mitosis and mitochondrial activity. Although functional studies have been performed on NSL complex members, none of the four core proteins, including Kansl3, have been studied during early mouse development. Here we show that homozygous knockout Kansl3 embryos are lethal at peri-implantation stages, failing to hatch out of the zona pellucida. When the zona pellucida is removed in vitro, Kansl3 null embryos form an abnormal outgrowth with significantly disrupted inner cell mass (ICM) morphology. We document lineage-specific defects at the blastocyst stage with significantly reduced ICM cell number but no difference in trophectoderm cell numbers. Both epiblast and primitive endoderm lineages are altered with reduced cell numbers in null mutants. These results show that Kansl3 is indispensable during early mouse embryonic development and with defects in both ICM and trophectoderm lineages.

Abstract 3055: Characterizing the role of HNF4α isoforms in human PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly and recalcitrant malignancy with a 5-year survival rate of 12%. Transcriptomic studies have classified PDAC into a classical or basal-like subtype. The classical subtype has a better prognosis, response to chemotherapy, and express higher levels of endodermal lineage specifiers compared to the basal-like subtype. However, the field lacks a comprehensive understanding of the key subtype-specific regulators. Functional analysis of transcription factors associated with each subtype are needed to elucidate their roles in regulation of PDAC subtypes and to identify therapeutically targetable vulnerabilities. Previous work in our lab demonstrated that HNF4α is a key regulator of the classical subtype. Using genetically engineered mouse models, our lab showed that loss of HNF4α leads to an increase in tumor burden, a shift from glandular to poorly differentiated histology, and a decrease in the classical subtype score. However, while murine PDAC only express one set of HNF4α isoforms, human PDAC express both sets of HNF4α isoforms, suggesting murine PDAC may not fully recapitulate human disease. There are 2 subsets of HNF4α isoforms driven by expression from 2 distinct promoters, P1 and P2. P1 and P2 isoforms are differentially expressed within the gastrointestinal (GI) tract and play dichotomous roles in GI malignancies. In HNF4α-positive human PDAC, P2 isoforms are always expressed but there is variable expression of P1 isoforms. Based on the differential role of HNF4α isoforms in both normal tissue and cancer, we hypothesize that P1 isoforms will restrain tumor growth and promote greater classical/GI differentiation compared to P2 isoforms. We utilized CRISPRi to selectively inhibit transcription at the P1 or P2 promoter in HNF4α-positive, human cell lines expressing both isoforms. To test for sufficiency, we exogenously expressed P1 or P2 isoforms in HNF4α-negative, human cell lines. We then performed Incucyte, CRISPRi competition, and anchorage-independent growth assays. We performed RNA-seq and ChIP-seq on both exogenous and endogenous HNF4α isoforms. Functional assays revealed that HNF4α isoforms regulated growth in a context dependent manner. Exogenous P1 isoforms significantly inhibited growth while loss of endogenous P1 isoforms was dispensable for growth. Exogenous P2 isoforms inhibited growth to a lesser degree while loss of endogenous P2 isoforms led to a significant reduction in growth. Despite ChIP-seq analysis revealing very similar binding profiles, RNAseq analysis revealed that P1 isoforms are significantly more transcriptionally active than P2 isoforms and are stronger inducers of GI differentiation. Taken together, our data demonstrates that P2 isoforms are more compatible with growth in PDAC compared to P1 isoforms, which promote a more differentiated cell state. Mechanistically, this may be due to the increased transcriptional activity of P1 isoforms. Citation Format: Pengshu Fang, Acramul Kabir, Gabriela Fort, Eric L. Snyder. Characterizing the role of HNF4α isoforms in human PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3055.

Abstract 5445: Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach

Abstract Introduction: The cancer stem cell hypothesis explains the heterogeneity, drug resistance, and metastatic potential of Small Cell Lung Cancer (SCLC). To address limited surgical tissue availability, we developed an induced pluripotent stem cell (iPSC) model for investigating SCLC stemness mechanisms and potential novel therapies. Cigarette smoke condensates (CSC) have previously been linked to stem cell activation in lung epithelial cells. We aimed to transform normal lung iPSCs (Lu-iPSC) through prolonged CSC exposure to identify candidate genes in the transition to cancer stem cells. Methods: Four Lu-iPSC clones derived from normal human small airway epithelial cells (SAEC) of two healthy donors were exposed to CSC at different concentrations for short- (7 days) and long-term (6 months) periods. RNA-seq was performed to identify differentially expressed genes (DEGs) relative to respective SAEC. Gene expression data of 23 SCLC cell lines and 38 NSCLC from the public databases was extracted and analyzed relative to SAEC. In vitro tri-lineage differentiation studies, drug sensitivity assays, and phenotypic characterization by flow cytometry was performed in this study. Results: Gene expression profiles of Lu-iPSCs closely resembled those of SCLC rather than NSCLC. Short-term CSC exposure reduced Lu-iPSC proliferation while increasing the expression of key stemness markers such as SALL1 in a dose-dependent manner. Concurrently, CSC exposure downregulated the expression of nodal and its inhibitors, LEFTY1 and LEFTY2, which can be considered as the earliest signs of differentiation events in Lu-iPSC. Long term CSC exposure induced expression of epithelial mesenchymal transition markers including ZEB1 and ZEB2. In-vitro trilineage differentiation studies revealed that CSC particularly decreased expression of endodermal lineage specific markers (GATA4, SOX17, and FOXA2) without affecting mesodermal and ectodermal markers. Importantly, CSC induced the phenotypic expression of several lung cancer stem cell markers (CD44, CD133, and CD338) along with resistance to cisplatin and topotecan. RNA seq analysis of long-term CSC exposed Lu-iPSCs identified 92 common DEGs (53 up and 39 down) relative to their controls. Gene set enrichment analysis on DEGs indicated upregulation of Notch signaling, IGFBPs, TGF beta receptor signaling, and NCAM1 interactions while GABA receptor activation and GPCR signaling were downregulated. Comprehensive RNA seq analysis of CSC exposed and unexposed Lu-iPSCs relative to SCLC and NSCLC, identified potential candidate genes associated with SCLC stemness. Conclusion: Our findings suggest that this iPSC model shows promise for investigating mechanisms mediating stemness in SCLC and identifying candidate genes and their associated pathways as potential targets for preclinical development of novel treatment regimens for these highly lethal neoplasms. Citation Format: Sudheer Kumar Gara, Haitao Wang, Anand Singh, Tuana Tolunay, Vivek Shukla, Ruihong Wang, Mary Zhang, Stephanie Shiffka, David S. Schrump. Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5445.

Abstract 3052: Investigating the role of FOXA1/2 & HNF4⍺ in pancreatic ductal adenocarcinoma

Abstract PDAC represents a formidable challenge with an alarmingly low 5-year survival rate of less than 12%. Integrated genomic, transcriptomic, and proteomic analyses have identified two distinct PDAC subtypes: classical and basal. The critical difference between these subtypes lies in the expression of endodermal lineage specifiers, with the classical subtype expressing FOXA1, FOXA2 (FOXA1/2), and HNF4⍺, critical for pancreatic cell-fate determination, while the basal subtype downregulates these genes, leading to the loss of endodermal identity. We reason that the transcription factors (TF), FOXA1/2 and HNF4α, that play critical roles in pancreatic development, are not only biomarkers of the classical subtype but are essential regulators of PDAC subtype differentiation state. Despite their relevance in other cancers, their specific functions in PDAC remain largely unexplored. We aim to define the functional roles of FOXA1/2 and HNF4⍺ in the classical PDAC subtype, exploring their regulation of growth and cellular identity. Previous published work in our lab has shown that exogenous HNF4⍺ restrains growth in a panel of human and murine PDAC cell lines. Furthermore, we have shown that HNF4⍺ regulates the classical subtype program of PDAC. While FOXA1/2 and HNF4⍺ cooperatively enact gene expression changes in normal tissue, the extent to which cooperative interactions between these TFs are required to activate the classical gene expression program of PDAC remains unknown. Overall, we will test the central hypothesis that FOXA1/2 and HNF4⍺ are critical regulators of the transcriptional network governing the cellular identity of classical PDAC. To test this hypothesis, we will be reconstituting and modulating the expression of these factors in human and murine PDAC cell and organoid lines. We will assess gene expression changes through RNA-seq and genomic localization of these TFs through ChIP-seq. We have conducted bulk RNA sequencing on Foxa1/2-positive and negative organoids to transcriptionally identify differences brought on by Foxa1/2 deletion and found over 2,200 differentially expressed genes. Foxa1/2 deletion led to a significant decline in the classical PDAC signature based on gene ontology. Furthermore, we show that HNF4α can inhibit organoid growth in the absence of FOXA1/2. We discovered that HNF4⍺ targets, such as Lgals4, expression levels are unaltered at the RNA level independent of FOXA1/2. By assessing the roles of FOXA1/2 and HNF4α in PDAC, we aim to determine the transcriptional network governed by these factors in the classical subtype of PDAC Citation Format: Walter A. Orellana, Katherine Gillis, Pengshu Fang, Gabriela Fort, Eric L. Snyder. Investigating the role of FOXA1/2 & HNF4⍺ in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3052.

Assessment of human embryonic stem cells differentiation into definitive endoderm lineage on the soft substrates.

Pluripotent stem cells (PSCs) hold enormous potential for treating multiple diseases owing to their ability to self-renew and differentiate into any cell type. Albeit possessing such promising potential, controlling their differentiation into a desired cell type continues to be a challenge. Recent studies suggest that PSCs respond to different substrate stiffness and, therefore, can differentiate towards some lineages via Hippo pathway. Human PSCs can also differentiate and self-organize into functional cells, such as organoids. Traditionally, human PSCs are differentiated on stiff plastic or glass plates towards definitive endoderm and then into functional pancreatic progenitor cells in the presence of soluble growth factors. Thus, whether stiffness plays any role in differentiation towards definitive endoderm from human pluripotent stem cells(hPSCs) remains unclear. Our study found that the directed differentiation of human embryonic stem cells towards endodermal lineage on the varying stiffness did not differ from the differentiation on stiff plastic dishes. We also observed no statistical difference between the expression of yes-associated protein(YAP) and phosphorylatedYAP. Furthermore, we demonstrate that lysophosphatidic acid, a YAP activator, enhanced definitive endoderm formation, whereas verteporfin, a YAP inhibitor, did not have the significant effect on the differentiation. In summary, our results suggest that human embryonic stem cells may not differentiate in response to changes in stiffness, and that such cues may not have as significant impact on the level of YAP. Our findings indicate that more research is needed to understand the direct relationship between biophysical forces and hPSCs differentiation.

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.

P157 Differentiation of Tonsil-derived mesenchymal stem cells to intestinal stem cells-like cells for cell therapy of inflammatory bowel disease

Abstract Background Tonsil-derived mesenchymal stem cells (TMSCs) were found to exhibit a faster growth rate compared to mesenchymal stem cells derived from other tissues. This is attributed to the relatively younger age of the tonsil tissue donors. Moreover, it has been verified that TMSCs possess the capability to undergo differentiation into endoderm lineages, giving rise to cell types like parathyroid glands and insulin-producing cells. In this study, we explored the possibility of differentiation of TMSCs into intestinal stem cells-like cells (ISC-like cells), a cell type of endoderm lineages for utilizing these cells for therapeutic applications in inflammatory bowel disease (IBD). Methods TMSCs were cultivated in DMEM (10% FBS) and used in the experiment when 90-100% confluency was observed. Adipose-derived mesenchymal stem cells (AMSCs) were used as the control for assessing the differentiation efficacy of TMSCs. The morphology of cells was verified at each differentiation stage, and the expression of endoderm and intestinal stem cell markers was confirmed using real-time PCR (qPCR), western blotting, and immunofluorescence (IF) staining. Results Subjected to 100ng/ml of Activin A for endoderm differentiation and cultured for 2 to 5 days, TMSCs exhibited an upregulation in endoderm markers (SOX17, FOXA2), as confirmed by real-time PCR. Specifically, under the influence of Activin A at 100ng/ml and cultured for 5 days, there was a significant increase in the expression of SOX17 (pre-differentiation vs. endoderm-differentiation, 0.999 vs. 3.610, P=0.0394) and FOXA2 (1.047 vs. 3.912, P=0.0297). Following the endoderm differentiation phase, the medium was changed to include 500ng/ml of FGF4 and 500ng/ml of Wnt3a for a 7-day period. Post-culture, the expression level of Lgr5, an intestinal stem cell (ISC) marker, was significantly increased in TMSCs (1.061 vs. 134.0, P<0.001). Notably, AMSCs displayed a lower expression rate compared to TMSCs (TMSCs vs. AMSCs, 134.0 vs. 9.819, P<0.001). Moreover, the spheroids formation was observed 7 days after the initiation of FGF4 and Wnt3a treatment, a phenomenon absent in AMSCs differentiation. Immunofluorescence staining for differentiation markers further validated these findings: the expression of SOX17, an endoderm marker, and the fluorescence density of LGR5, an ISC marker, were notably higher in TMSCs compared to AMSCs. Protein expression levels also confirmed the upregulation of each marker in TMSCs. Conclusion This study validated the potential of TMSCs to differentiate into ISC-like cells, with a higher efficiency observed compared to AMSCs. The prospect of differentiating into ISC positions TMSCs as promising candidates for use as a tool in IBD.

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.

Models of fibrolamellar carcinomas, tools for evaluation of a new era of treatments.

Fibrolamellar carcinoma (FLC) is a rare but fatal cancer that occurs primarily in young people. There are currently no known effective treatments, although several promising treatments appear to be in development. Genetic studies have confirmed that almost all FLC tumors have a fusion protein marker (DNAJB1-PRKACA) encoded by a fusion gene (DNAJB1-PRKACA); It is currently accepted as a diagnostic criterion for FLCs. Several research teams have established patient-derived xenograft (PDX) FLC models using immunocompromised animals as hosts and patient tissue samples (tumors or ascites) as primary sources for PDX-derived organoids. These FLC organoids are composed of FLC epithelia, endothelial progenitor cells, and stellate cells. CRISPR/Cas9 was used as a gene editing technique to modify mature hepatocytes to obtain ex vivo FLC-like cells expressing the fusion gene and/or other mutated genes associated with FLCs. Although these models simulate some but not all FLC features. Drug screening using these models has not proven effective in identifying clinically useful treatments. Genetic studies comparing FLCs to normal maturing endodermal cell lineages have shown that FLCs share genetic signatures not with hepatocytes, but with subpopulations of biliary tree stem cells (BTSCs), hepato/pancreatic stem/progenitor cells that consistently reside in peribiliary glands (PBGs) located in the biliary tree and are sources of stem cells for the formation and postnatal regeneration of the liver and pancreas. Therefore, it is expected that models of BTSCs, instead of hepatocytes may prove more useful. In this review, we summarize the status of the various FLC models and their features, applications, and limitations. They provide opportunities to understand the cause and characteristics of this deadly disease and are models from which effective treatments can be identified.

Evidence for Functional Roles of MicroRNAs in Lineage Specification During Mouse and Human Preimplantation Development.

Proper formation of the blastocyst, including the specification of the first embryonic cellular lineages, is required to ensure healthy embryo development and can significantly impact the success of assisted reproductive technologies (ARTs). However, the regulatory role of microRNAs in early development, particularly in the context of preimplantation lineage specification, remains largely unknown. Taking a cross-species approach, this review aims to summarize the expression dynamics and functional significance of microRNAs in the differentiation and maintenance of lineage identity in both the mouse and the human. Findings are consolidated from studies conducted using in vitro embryonic stem cell models representing the epiblast, trophectoderm, and primitive endoderm lineages (modeled by naïve embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells, respectively) to provide insight on what may be occurring in the embryo. Additionally, studies directly conducted in both mouse and human embryos are discussed, emphasizing similarities to the stem cell models and the gaps in our understanding, which will hopefully lead to further investigation of these areas. By unraveling the intricate mechanisms by which microRNAs regulate the specification and maintenance of cellular lineages in the blastocyst, we can leverage this knowledge to further optimize stem cell-based models such as the blastoids, enhance embryo competence, and develop methods of non-invasive embryo selection, which can potentially increase the success rates of assisted reproductive technologies and improve the experiences of those receiving fertility treatments.

Human RFX6 regulates endoderm patterning at the primitive gut tube stage.

Transcriptional factor RFX6 is known to be a causal gene of Mitchell-Riley syndrome (MRS), an autosomal recessive neonatal diabetes associated with pancreatic hypoplasia and intestinal atresia/malformation. The morphological defects are limited to posterior foregut and mid-hindgut endodermal lineages and do not occur in the anterior foregut lineage; the mechanism remains to be fully elucidated. In this study, we generated RFX6+/eGFP heterozygous knockin and RFX6eGFP/eGFP homozygous knockin/knockout human-induced pluripotent stem cell (hiPSC) lines and performed in vitro endoderm differentiation to clarify the role of RFX6 in early endoderm development. RFX6 expression was found to surge at the primitive gut tube (PGT) stage in comparison with that in the undifferentiated or definitive endoderm stage. At the PGT stage, the expression of PDX1 and CDX2, posterior foregut and mid-hindgut master regulators, respectively, was decreased by the RFX6 deficit. PDX1+ and CDX2+ cells were mostly green fluorescent protein (GFP)+ in RFX6+/eGFP hiPSCs, but their cell number was markedly decreased in RFX6eGFP/eGFP hiPSCs. The expression of SOX2, an anterior foregut marker, was not affected by the RFX6 deficit. In addition, we found a putative RFX6-binding X-box motif using cap analysis of gene expression-seq and the motif-containing sequences in the enhancer regions of PDX1 and CDX2 bound to RFX6 in vitro. Thus, RFX6 regulates the ParaHox genes PDX1 and CDX2 but does not affect SOX2 in early endodermal differentiation, suggesting that defects in early stage endoderm patterning account for the morphological pathology of MRS.

Branchioma: immunohistochemical and molecular genetic study of 23 cases highlighting frequent loss of retinoblastoma 1 immunoexpression.

Branchioma is an uncommon benign neoplasm with an adult male predominance, typically occurring in the lower neck region. Different names have been used for this entity in the past (ectopic hamartomatous thymoma, branchial anlage mixed tumor, thymic anlage tumor, biphenotypic branchioma), but currently, the term branchioma has been widely accepted. Branchioma is composed of endodermal and mesodermal lineage derivatives, in particular epithelial islands, spindle cells, and mature adipose tissue without preexistent thymic tissue or evidence of thymic differentiation. Twenty-three branchiomas were evaluated morphologically. Eighteen cases with sufficient tissue were assessed by immunohistochemistry, next-generation sequencing (NGS) using the Illumina Oncology TS500 panel, and fluorescence in situ hybridization (FISH) using an RB1 dual-color probe. All cases showed a biphasic morphology of epithelial and spindle cells with intermingled fatty tissue. Carcinoma arising in branchioma was detected in three cases. The neoplastic cells showed strong AE1/3 immunolabeling (100%), while the spindle cells expressed CD34, p63, and SMA (100%); AR was detected in 40-100% of nuclei (mean, 47%) in 14 cases. Rb1 showed nuclear loss in ≥ 95% of neoplastic cells in 16 cases (89%), while two cases revealed retained expression in 10-20% of tumor cell nuclei. NGS revealed a variable spectrum of likely pathogenic variants (n = 5) or variants of unknown clinical significance (n = 6). Loss of Rb1 was detected by FISH in two cases. Recent developments support branchioma as a true neoplasm, most likely derived from the rudimental embryological structures of endoderm and mesoderm. Frequent Rb1 loss by immunohistochemistry and heterozygous deletion by FISH is a real pitfall and potential confusion with other Rb1-deficient head and neck neoplasms (i.e., spindle cell lipoma), especially in small biopsy specimens.

Time series single-cell transcriptional atlases reveal cell fate differentiation driven by light in Arabidopsis seedlings

Light serves as the energy source for plants as well as a signal for growth and development during their whole life cycle. Seedling de-etiolation is the most dramatic manifestation of light-regulated plant development processes, as massive reprogramming of the plant transcriptome occurs at this time. Although several studies have reported about organ-specific development and expression induced by light, a systematic analysis of cell-type-specific differentiation and the associated transcriptional regulation is still lacking. Here we obtained single-cell transcriptional atlases for etiolated, de-etiolating and light-grown Arabidopsisthaliana seedlings. Informative cells from shoot and root tissues were grouped into 48 different cell clusters and finely annotated using multiple markers. With the determination of comprehensive developmental trajectories, we demonstrate light modulation of cell fate determination during guard cell specialization and vasculature development. Comparison of expression atlases between wild type and the pifq mutant indicates that phytochrome-interacting factors (PIFs) are involved in distinct developmental processes in endodermal and stomatal lineage cells via controlling cell-type-specific expression of target genes. These results provide information concerning the light signalling networks at the cell-type resolution, improving our understanding of how light regulates plant development at the cell-type and genome-wide levels. The obtained information could serve as a valuable resource for comprehensively investigating the molecular mechanism of cell development and differentiation in response to light.

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.

Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells

BackgroundIn vitro production of hematopoietic stem/progenitor cells (HSPCs) from human-induced pluripotent stem cells (hiPSCs) provides opportunities for fundamental research, disease modeling, and large-scale production of HLA-matched HSPCs for therapeutic applications. However, a comprehensive understanding of the signaling mechanisms that regulate human hematopoiesis is needed to develop a more effective procedure for deriving HSPCs from hiPSCs.MethodsIn this study, we investigate the role of YAP during the hematopoietic differentiation of hiPSCs to HSPCs and erythrocytes using the isogenic YAP-overexpressing (YAP-S5A) and YAP-depleting (YAP-KD) hiPSCs to eliminate the effects of a genetic background variation.ResultsAlthough YAP is dispensable for maintaining the self-renewal and pluripotency of these hiPSCs, it affects the early cell-fate determination and hematopoietic differentiation of hiPSCs. Depleting YAP enhances the derivation efficiency of HSPCs from hiPSCs by inducing the mesodermal lineage commitment, promoting hematopoietic differentiation, and preventing the differentiation toward endothelial lineage. On the contrary, the overexpression of YAP reduced HSPCs yield by inducing the endodermal lineage commitment, suppressing hematopoietic differentiation, and promoting the differentiation toward endothelial lineage.ConclusionsExpression of YAP is crucial for the differentiation of hiPSC-derived HSPCs toward mature erythrocytes. We believe that by manipulating YAP activity using small molecules, the efficiency of the large-scale in vitro production system for generating hematopoietic stem/progenitor cells for future therapeutic use could be improved.