Stem Cell Lineage Specification Research Articles

NFKBIA deletions reshape the epigenome antithetical to the IDH mutation and indication of prognosis in diffuse gliomas.

2083 Background: Genetic alterations help predict the clinical behavior of diffuse gliomas, but some variability remains uncorrelated. The NFKBIA gene at 14q13.2 encodes IkBa, a protein that regulates the activity of NF-kB in the cytoplasm. Evidence that, in chromatin, nuclear NFKBIA dynamically interacts with histones H2A and H4 to regulate polycomb-dependent transcriptional repression and, thus, stem cell maturation and lineage specification and our characterization of NFKBIA as a tumor suppressor in glioblastoma prompted our investigation of the relationship of NFKBIA deletions to other genetic markers, alterations in the methylome, and the clinical course of gliomas. Methods: We analyzed the genetic profiles of 2,343 WHO grade 2 to 4 diffuse gliomas in multiple clinically well-characterized patient populations, including national randomized phase III consortium trial RTOG 9802, to determine whether incorporation of NFKBIA deletions could enhance the prognostic value of current molecular descriptions. Results: We demonstrate that haploinsufficient deletions of NFKBIA display distinct patterns of occurrence in relation to other genetic markers, including driver mutations IDH, TERT, ATRX, and PTEN, 1p19q codeletion, and CDKN2A/B deletion, and are disproportionally present at recurrence. NFKBIA haploinsufficiency is associated with unfavorable patient outcomes, independent of genetic and clinicopathologic predictors. NFKBIA deletions reshape the DNA and histone methylome antipodal to the IDH mutation. The methylome changes facilitated through NFKBIA deletions engender a transcriptome landscape partly reminiscent of H3K27M mutant pediatric gliomas and are highly enriched for neural stem cell and neurogenesis genes bearing repressive H3K27 marks. Our findings imply that the NFKBIA deletion in adult gliomas and the H3K27M mutation in pediatric diffuse midline gliomas define aggressive disease subtypes that may share an epigenetic state reflecting a common PRC2 loss-of-function phenotype antipodal to the quasi PRC2 gain-of-function phenotype of IDH mutant gliomas and their favorable clinical course. Lower-grade gliomas harboring NFKBIA deletions behave much like high-grade gliomas. In IDH mutant gliomas, NFKBIA deletions are common in tumors with a clinical course similar to that of IDH wildtype tumors. A sparse, easily interpretable, and externally validated nomogram model for estimating individual patient survival in IDH mutant gliomas, incorporating NFKBIA deletion and current best-established genetic and clinicopathologic factors, confirms that NFKBIA deletions predict comparatively brief survival. Conclusions: NFKBIA haploinsufficiency aligns with distinct epigenome changes, portends a poor prognosis, and should be incorporated into clinical models predicting the disease fate of diffuse gliomas.

Histone demethylase KDM3B mediates matrix stiffness-induced osteogenic differentiation of adipose-derived stem cells

Biomechanical signals in the extracellular niche are considered promising for programming the lineage specification of stem cells. Recent studies have reported that biomechanics, such as the microstructure of nanomaterials, can induce adipose-derived stem cells (ASCs) to differentiate into osteoblasts, mediating gene regulation at the epigenetic level. Therefore, in this study, transcriptome expression levels of histone demethylases in ASCs were screened after treatment with different matrix stiffnesses, and histone lysine demethylase 3B (KDM3B) was found to promote osteogenic differentiation of ASCs in response to matrix stiffness, indicating a positive modulatory effect on this biological process. ASCs exhibited widespread and polygonal shapes with a distinct bundle-like expression of vinculin parallel to the axial cytoskeleton along the cell margins on the stiff matrix rather than round shapes with a smeared and shorter expression on the soft matrix. Comparatively rigid polydimethylsiloxane material directed ASCs into an osteogenic phenotype in inductive culture media via the upregulation of osteocalcin, alkaline phosphatase, and runt-related transcription factor 2. Treatment with KDM3B-siRNA decreased the expression of osteogenic differentiation markers and impaired mitochondrial dynamics and mitochondrial membrane potential. These results illustrate the critical role of KDM3B in the biomechanics-induced osteogenic commitment of ASCs and provide new avenues for the further application of stem cells as potential therapeutics for bone regeneration.

Enhanced glucose metabolism in Tet-deficient mouse embryonic stem cells

Interactions between epigenetics and metabolites play critical roles in regulating the pluripotency and differentiation of embryonic stem cells. Proper glucose metabolism and DNA methylation are essential for orchestrating accurate lineage specification and the normal functions of embryonic stem cells. However, the impact of Ten-eleven Translocation (TET)-mediated DNA methylation modifications on the metabolism of mouse embryonic stem cells (mESCs) remains less well defined. In this study, we investigated the consequences of Tet triple knockout (Tet-TKO) in mESCs and observed notable alterations in glucose metabolism. These changes were marked by enhanced glucose uptake and glycolysis, likely owing to the upregulation of genes critical for glucose metabolism. Furthermore, Tet-TKO mESCs exhibited defects in glucose-dependent differentiation, suggesting that cells with epigenetic defects might display metabolic vulnerability when exposed to external nutritional cues. Collectively, our findings establish the pivotal role of the TET family of dioxygenases in maintaining proper glucose metabolism and safeguarding stem cell lineage specification, thus enhancing our understanding of the intricate interplay between epigenetic modifications and cellular metabolism in stem cells.

Localized compaction of collagen hydrogels using acoustic droplet vaporization promotes osteogenic differentiation of mesenchymal stem cells

The lineage specification of mesenchymal stem cells (MSCs) is dependent on matrix stiffness, with osteogenesis promoted on or within stiff microenvironments. Unlike native extracellular matrix, conventional hydrogels have relatively static and uniform mechanical properties, thereby limiting the study of how spatiotemporally controlled mechanical cues impact MSC behavior. Here, we spatiotemporally control MSC differentiation in 3D hydrogels by actively modulating matrix stiffness using acoustic droplet vaporization (ADV). An acoustically responsive scaffold (ARS) was generated by co-encapsulating MSCs and perfluorohexane-based emulsion (13 μm) within type I collagen. ADV was used to generate bubbles within the ARS. Bubble diameter and the width of the compacted matrix region increased over time following ADV. Atomic force microscopy measurements demonstrated that the hydrogel region proximal to the bubble was significantly stiffer than the distal regions. Significantly greater levels of Runx2 and osteocalcin expression were observed in MSCs proximal to bubbles compared to distal on days 7 and 14. Higher alkaline phosphatase activity also validated the above findings. The significant upregulation of these osteogenic biomarkers suggests ADV-induced, mechanical changes in ARSs were sufficient to enhance osteogenic differentiation of MSCs. This approach is a significant step towards controlling the 3D differentiation of MSCs in a spatially localized, non-invasive, and on-demand manner.

Genetic profiling of azoospermic men to identify the etiology and predict reproductive potential

PurposeTo identify germline mutations related to azoospermia etiology and reproductive potential of surgically retrieved spermatozoa, and to investigate the feasibility of predicting seminiferous tubule function of nonobstructive azoospermic men by transcriptomic profiling of ejaculates.Materials and methodsSperm specimens were obtained from 30 men (38.4 ± 6 years) undergoing epididymal sperm aspiration for obstructive azoospermia (OA, n = 19) acquired by vasectomy, or testicular biopsy for nonobstructive azoospermia (NOA, n = 11). To evaluate for a correlation with azoospermia etiology, DNAseq was performed on surgically retrieved spermatozoa, and cell-free RNAseq on seminal fluid (n = 23) was performed to predict spermatogenesis in the seminiferous tubule.ResultsOverall, surgically retrieved sperm aneuploidy rates were 1.7% and 1.8% among OA and NOA cohorts, respectively. OA men carried housekeeping-related gene mutations, while NOA men displayed mutations on genes involved in crucial spermiogenic functions (AP1S2, AP1G2, APOE). We categorized couples within each cohort according to ICSI clinical outcomes to investigate genetic causes that may affect reproductive potential. All OA-fertile men (n = 9) carried mutations in ZNF749 (sperm production), whereas OA-infertile men (n = 10) harbored mutations in PRB1, which is essential for DNA replication. NOA-fertile men (n = 8) carried mutations in MPIG6B (stem cell lineage differentiation), whereas NOA-infertile individuals (n = 3) harbored mutations in genes involved in spermato/spermio-genesis (ADAM29, SPATA31E1, MAK, POLG, IFT43, ATG9B) and early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Transcriptomic assessment of cell-free RNAs in seminal fluid from NOA men allowed the prediction of residual spermatogenic foci.ConclusionsSperm genome profiling provides invaluable information on azoospermia etiology and identifies gene-related mechanistic links to reproductive performance. Moreover, RNAseq assessment of seminal fluid from NOA men can help predict sperm retrieval during testicular biopsies.

Metabolic regulation of erythrocyte development and disorders

The formation of new red blood cells (RBC) (erythropoiesis) has served as a paradigm for understanding cellular differentiation and developmental control of gene expression. The metabolic regulation of this complex, coordinated process remains poorly understood. Each step of erythropoiesis, including lineage specification of hematopoietic stem cells, proliferation, differentiation, and terminal maturation into highly specialized oxygen-carrying cells, has unique metabolic requirements. Developing erythrocytes in mammals are also characterized by unique metabolic events such as loss of mitochondria with switch to glycolysis, ejection of nucleus and organelles, high-level heme and hemoglobin synthesis, and antioxidant requirement to protect hemoglobin molecules. Genetic defects in metabolic enzymes, including pyruvate kinase and glucose-6-phosphate dehydrogenase, cause common erythrocyte disorders, whereas other inherited disorders such as sickle cell disease and β-thalassemia display metabolic abnormalities associated with disease pathophysiology. Here we describe recent discoveries on the metabolic control of RBC formation and function, highlight emerging concepts in understanding the erythroid metabolome, and discuss potential therapeutic benefits of targeting metabolism for RBC disorders.

Growth Factor Independence-1 (GFI-1) Gene Expression in Hematopoietic Stem Cell Lineage Differentiation in Low Birth Weight Newborns Compared With Normal Birth Weight Newborns at Term Pregnancy.

Introduction Low birth weight (LBW), which is a risk factor for noncommunicable diseases throughout life, is a significant public health concern. In addition to regulating myeloid cell differentiation and proliferation, a transcriptional repressor identified as growth factor independence-1 (GFI-1) is essential for hematopoietic stem cell maintenance and self-renewal. The current study was designed to compare the expression of the GFI-1 gene in the differentiation of hematopoietic stem cells in newborns with LBW and those with normal birth weight (NBW). Methods A prospective comparative analytical study was carried out from September 2019 to September 2021 after obtaining Institute Ethical Committee approval at a tertiary care center in north India. The GFI-1 gene expression levels in 50 cord blood samples from women with term gestation and LBW newborns (<2500 grams) were measured using quantitative real-time polymerase chain reaction (RT-PCR) and compared to gene expression levels in 50 cord blood samples from women with term gestation and NBW newborns (≥2500 grams). The data were analyzed using IBM SPSS statistics software version 24.0 (IBM Corp., Armonk, NY). Results The median GFI-1 expression in LBW newborns is 3.1, whereas among NBW newborns it is 9.39. The difference is significant (P <0.001). The level of GFI-1 gene expression in LBW newborns was correlated with their birth weight. The coefficient of correlation was found to be weakly positive (r = 0.223). The birth weight of NBW newborns was correlated to the level of expression of the GFI-1 gene, which was found to be positively correlated (r = 0.332). Conclusion The levels of the GFI-1 gene and newborn birth weight were compared in LBW infants, which were weakly positively correlated. The level of GFI-1 gene expression at birth was compared to the birth weight of NBW newborns, which was positively correlated.

Stomatal cell fate commitment via transcriptional and epigenetic control: Timing is crucial.

The formation of stomata presents a compelling model system for comprehending the initiation, proliferation, commitmentand differentiation of de novo lineage-specific stem cells. Precise, timelyand robust cell fate and identity decisions are crucial for the proper progression and differentiation of functional stomata. Deviations from this precise specification result in developmental abnormalities and nonfunctional stomata. However, the molecular underpinnings of timely cell fate commitment have just begun to be unravelled. In this review, we explore the key regulatory strategies governing cell fate commitment, emphasizing the distinctions between embryonic and postembryonic stomatal development. Furthermore, the interplay of transcription factors and cell cycle machineries is pivotal in specifying the transition into differentiation. We aim to synthesize recent studies utilizing single-cell as well as cell-type-specific transcriptomics, epigenomicsand chromatin accessibility profiling to shed light on how master-regulatory transcription factors and epigenetic machineries mutually influence each other to drive fate commitment and maintenance.

Metabolic Remodeling during Early Cardiac Lineage Specification of Pluripotent Stem Cells.

Growing evidence indicates that metabolites and energy metabolism play an active rather than consequential role in regulating cellular fate. Cardiac development requires dramatic metabolic remodeling from relying primarily on glycolysis in pluripotent stem cells (PSCs) to oxidizing a wide array of energy substrates to match the high bioenergetic demands of continuous contraction in the developed heart. However, a detailed analysis of how remodeling of energy metabolism contributes to human cardiac development is lacking. Using dynamic multiple reaction monitoring metabolomics of central carbon metabolism, we evaluated temporal changes in energy metabolism during human PSC 3D cardiac lineage specification. Significant metabolic remodeling occurs during the complete differentiation, yet temporal analysis revealed that most changes occur during transitions from pluripotency to mesoderm (day 1) and mesoderm to early cardiac (day 5), with limited maturation of cardiac metabolism beyond day 5. Real-time metabolic analysis demonstrated that while hPSC cardiomyocytes (hPSC-CM) showed elevated rates of oxidative metabolism compared to PSCs, they still retained high glycolytic rates, confirming an immature metabolic phenotype. These observations support the opportunity to metabolically optimize the differentiation process to support lineage specification and maturation of hPSC-CMs.

Basement Membrane Mimetic Hydrogel Cooperates with Rho-Associated Protein Kinase Inhibitor to Promote the Development of Acini-Like Salivary Gland Spheroids.

Successful engineering of functional salivary glands necessitates the creation of cell-instructive environments for ex vivo expansion and lineage specification of primary human salivary gland stem cells (hS/PCs). Herein, basement membrane mimetic hydrogels were prepared using hyaluronic acid, cell adhesive peptides, and hyperbranched polyglycerol (HPG), with or without sulfate groups, to produce "hyperGel+" or "hyperGel", respectively. Differential scanning fluorescence experiments confirmed the ability of the sulphated HPG precursor to stabilize fibroblast growth factor 10. The hydrogels were nanoporous, cytocompatibile and cell-permissive, enabling the development of multicellular hS/PC spheroids in 14 days. Incorporation of sulfated HPG species in the hydrogel enhanced cell proliferation. Culture of hS/PCs in hyperGel+ in the presence of a Rho kinase inhibitor, Y-27632 (Y-27), led to the development of spheroids with a central lumen, increased the expression of acinar marker aquaporin-3 at the transcript level (AQP3), and decreased the expression of ductal marker keratin 7 at both the transcript (KRT7) and the protein levels (K7). Reduced expression of transforming growth factor beta (TGF-β) targets SMAD2/3 was also observed in Y27-treated cultures, suggesting attenuation of TGF-β signaling. Thus, hyperGel+ cooperates with the ROCK inhibitor to promote the development of lumened spheroids with enhanced expression of acinar markers.

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.

The matrix microenvironment influences but does not dominate tissue-specific stem cell lineage differentiation

Mesenchymal stem cells (MSCs) play a pivotal role in tissue engineering and regenerative medicine, with their clinical application often hindered by cell senescence during ex vivo expansion. Recent studies suggest that MSC-deposited decellularized extracellular matrix (dECM) offers a conducive microenvironment that fosters cell proliferation and accentuates stem cell differentiation. However, the ability of this matrix environment to govern lineage differentiation of tissue-specific stem cells remains ambiguous. This research employs human adipose-derived MSCs (ADSCs) and synovium-derived MSCs (SDSCs) as models for adipogenesis and chondrogenesis differentiation pathways, respectively. Genetically modified dECM (GMdECM), produced by SV40LT-transduced immortalized cells, was studied for its influence on cell differentiation. Both types of immortalized cells displayed a reduction in chondrogenic ability but an enhancement in adipogenic potential. ADSCs grown on ADSC-deposited dECM showed stable chondrogenic potential but increased adipogenic capacity; conversely, SDSCs expanded on SDSC-generated dECM displayed elevated chondrogenic capacity and diminished adipogenic potential. This cell-dependent response was confirmed through GMdECM expansion, with SDSCs showing enhanced chondrogenesis. However, ADSCs did not exhibit improved chondrogenic potential on GMdECM, suggesting that the matrix microenvironment does not dictate the final differentiation path of tissue-specific stem cells. Potential molecular mechanisms, such as elevated basement membrane protein expression in GMdECMs and dynamic TWIST1 expression during expansion and chondrogenic induction, may underpin the strong chondrogenic differentiation of GMdECM-expanded SDSCs.

Intracellular pH dynamics regulates intestinal stem cell lineage specification

Intracellular pH dynamics is increasingly recognized to regulate myriad cell behaviors. We report a finding that intracellular pH dynamics also regulates adult stem cell lineage specification. We identify an intracellular pH gradient in mouse small intestinal crypts, lowest in crypt stem cells and increasing along the crypt column. Disrupting this gradient by inhibiting H+ efflux by Na+/H+ exchanger 1 abolishes crypt budding and blocks differentiation of Paneth cells, which are rescued with exogenous WNT. Using single-cell RNA sequencing and lineage tracing we demonstrate that intracellular pH dynamics acts downstream of ATOH1, with increased pH promoting differentiation toward the secretory lineage. Our findings indicate that an increase in pH is required for the lineage specification that contributes to crypt maintenance, establishing a role for intracellular pH dynamics in cell fate decisions within an adult stem cell lineage.

P-712 Transcriptomic Prediction of Spermatogenesis in the Ejaculates of Nonobstructive Azoospermic Men and Genomic Characterization of their Embryo Developmental Competence

Abstract Study question Can the genomic assessment of ejaculates from azoospermic men predict successful sperm extraction at testicular biopsy (TESE) and provide information on their reproductive potential? Summary answer RNAseq on the ejaculates of NOA men identified transcriptomic predictors of successful TESE outcome, while DNAseq evidenced gene-related mechanistics linked to reproductive performance. What is known already The most puzzling form of azoospermia is the testicular type. The chances of successful surgical sperm retrieval for these cases can be unpredictable, even when a benign histopathology has been obtained. The ability to identify individuals with complete loss of spermatogenesis at the seminiferous tubule level would help eliminate anesthesia and surgical risks, as well as financial and emotional distress. We propose to noninvasively assess the ejaculates from these men to predict successful TESE sperm retrieval. We utilize DNAseq to assess the impact of germline mutations on the embryo developmental competence of the surgically retrieved gametes. Study design, size, duration Over 4 years, we recruited men with normal peripheral karyotypes and azoospermia (n = 23), confirmed by absence of spermatozoa in extensive semen analyses. We performed epigenetic assessments on their ejaculates to compare transcriptomic profiles according to whether spermatozoa were successfully retrieved after TESE ((+)Sperm,n=11), or not ((-)Sperm,n=12). We then performed DNAseq (IRB 1006101085) on the testicular spermatozoa retrieved, to assess their ability to generate a pregnancy (fertile) or not (infertile), while controlling for maternal age. Participants/materials, setting, methods RNA was isolated from seminal fluid for epigenetic assessment (21,855 genes). A &amp;gt; 1 absolute log2fold change and P&amp;lt;0.0005 were considered significant. Transcriptomic profiles were compared between the (-)Sperm and (+)Sperm cohorts. For genetic assessment, surgically retrieved spermatozoal DNA from the (+)Sperm and obstructive azoospermic (OA) control (n = 19) cohorts were extracted and amplified. Gene mutation profiles, detected by CLC Genomic Server 9.0, were assessed for the (+)Sperm cohort according to ICSI outcomes, compared to the OA-control. Main results and the role of chance Transcriptomic analyses on seminal plasma from the 23 NOA men identified 8 imbalanced genes, involved in spermatogenesis (TMCO5B,C10orf62,SMKR1,SPZ1), sperm function (NEU1,TPTE2), and testis development (TRPC1,IGSF11-AS1). TPTE2 was almost completely expressed in 81.8%(9/11) of the (+)Sperm cohort (paternal age, 37.1±6yrs), while IGSF11-AS1 was underexpressed in all 12 (-)Sperm men (paternal age, 34.3±5yrs). Both genes are implicated in spermatogenic defects and are normally highly expressed in testis. Interestingly, NEU1, localized on the sperm acrosome and crucial for sperm capacitation, was exclusively underexpressed in (-)Sperm while overexpressed in (+)Sperm cohorts. Genomic analyses of spermatozoal DNA from the (+)Sperm cohort was compared to a matched control of OA patients (paternal age, 36.8±7yrs). Spermatozoa from the study cohort displayed mutations on genes involved in RNA transcription (POLR2L), apoptosis (AP5M1), and spermiogenic functions (AP1S2,AP1G2,APOE), while these genes were unaffected in the OA-control. When we compared sperm genetic profiles of the successful TESE cohort (maternal age, 36.8±2yrs, paternal age, 37.1±6yrs) according to ICSI outcome, we found that spermatozoa from the 8 fertile patients, all with term pregnancies, displayed consistent mild mutations on MPIG6B (stem cell lineage differentiation) while gametes from their 3 infertile counterparts carried severe mutations on genes (MBD5,CCAR1,PMEPA1,POLK,REC8,REPIN1,MAPRE3,ARL4C) involved in early embryonic development, and therefore failed to achieve embryo implantation. Limitations, reasons for caution Although we predicted, by transcriptomic analyses of ejaculates from NOA men, the presence of spermatozoa and characterized their embryo developmental competence through genetic profiling, these preliminary results need validation in a larger study cohort. Moreover, although controlled for, a confounding female factor cannot be undoubtfully excluded. Wider implications of the findings Epigenetic assessment of seminal fluid from NOA men can noninvasively identify biomarkers capable of predicting complete loss of spermatogenesis, sparing these patients from unnecessary surgery. Gene profiling can identify gametes capable of supporting term pregnancies, laying the groundwork for precision medicine in male infertility screening. Trial registration number n/a

Transforming Growth Factor BetaPromotes Inflammation and Tumorigenesis in Smad4-Deficient Intestinal Epithelium in a YAP-Dependent Manner.

Transforming growth factor beta (TGF-β), a multifunctional cytokine, plays critical roles in immune responses. However, the precise role of TGF-β in colitis and colitis-associated cancer remains poorly defined. Here, it is demonstrated that TGF-β promotes the colonic inflammation and related tumorigenesis in the absence of Smad family member 4 (Smad4). Smad4 loss in intestinal epithelium aggravates colitis and colitis-associated neoplasia induced by dextran sulfate sodium (DSS) and azoxymethane/dextran sulfate sodium (AOM/DSS), leading to over-activated immune responses and increased TGF-β1 levels. In Smad4-deficient organoids, TGF-β1 stimulates spheroid formation and impairs intestinal stem cell proliferation and lineage specification. YAP, whose expression is directly upregulated by TGF-β1 after Smad4 deletion, mediates the effect of TGF-β1 by interacting with Smad2/3. Attenuation of YAP/TAZ prevents TGF-β1-induced spheroid formation in Smad4-/- organoids and alleviates colitis and colitis-associated cancer in Smad4-deficient mice. Collectively, these results highlight an integral role of the TGF-β/Smad4 axis in restraining intestinal inflammation and tumorigenesis and suggest TGF-β or YAP signaling as therapeutic targets for these gastrointestinal diseases intervention.

3D genome perspective on cell fate determination, organ regeneration, and diseases

The nucleosome is the fundamental subunit of chromatin. Nucleosome structures are formed by the combination of histone octamers and genomic DNA. Through a systematic and precise process of folding and compression, these structures form a 30-nm chromatin fibre that is further organized within the nucleus in a hierarchical manner, known as the 3D genome. Understanding the intricacies of chromatin structure and the regulatory mode governing chromatin interactions is essential for unravelling the complexities of cellular architecture and function, particularly in relation to cell fate determination, regeneration, and the development of diseases. Here, we provide a general overview of the hierarchical structure of chromatin as well as of the evolution of chromatin conformation capture techniques. We also discuss the dynamic regulatory changes in higher-order chromatin structure that occur during stem cell lineage differentiation and somatic cell reprogramming, potential regulatory insights at the chromatin level in organ regeneration, and aberrant chromatin regulation in diseases.

Hierarchical hydrogel microarrays fabricated based on a microfluidic printing platform for high-throughput screening of stem cell lineage specification.

2D cell cultures are suitable for rapid exploration of the factors in the extracellular matrix affecting the development of cells. The technology of the micrometre-sized hydrogel array provides a feasible, miniaturized, and high-throughput strategy for the process. However, current microarray devices lack a handy and parallelized methodology in sample treatment, which makes the process of high-throughput cell screening (HTCS) expensive and inefficient. Here, based on the functionalization of micro-nano structures and the fluid control capability of microfluidic chips, we build a microfluidic spotting-screening platform (MSSP). The MSSP can print 20000 microdroplet spots within 5 min, coupled with a simple strategy for parallel addition of compound libraries. Compared with open microdroplet arrays, the MSSP can control the evaporation rate of nanoliter droplets, providing a stable fabrication platform for hydrogel-microarray-based materials. As a proof-of-concept demonstration, the MSSP successfully controlled the adhesion, adipogenic, and osteogenic differentiation behavior of mesenchymal stem cells by rationally designing the substrate stiffness, adhesion area, and cell density. We anticipate that the MSSP may provide an accessible and promising tool for hydrogel-based HTCS. STATEMENT OF SIGNIFICANCE: High-throughput screening of cells is a common approach to improve the efficiency of biological experiments, and one challenge of the existing technologies is to achieve rapid and precise cell screening with a low-cost and simple strategy. Through the integration of the microfluidic and micro-nanostructure technologies, we fabricated a microfluidic spotting-screening platforms. Benefiting from the flexible control of the fluids, the device can print 20000 microdroplet spots within 5 min, coupled with a simple procedure for parallel addition of compound libraries. High-throughput screening of stem cell lineage specification has also been achieved by the platform, which provides a high-throughput, high-content information extraction strategy for cell-biomaterial interaction research.

An Example of Neuro-Glial Commitment and Differentiation of Muse Stem Cells Obtained from Patients with IQSEC2-Related Neural Disorder: A Possible New Cell-Based Disease Model.

Although adult stem cells may be useful for studying tissue-specific diseases, they cannot be used as a general model for investigating human illnesses given their limited differentiation potential. Multilineage-differentiating stress-enduring (Muse) stem cells, a SSEA3(+) cell population isolated from mesenchymal stromal cells, fat, and skin fibroblasts, may be able to overcome that restriction. The Muse cells present in fibroblast cultures obtained from biopsies of patients' skin may be differentiated into cells of interest for analyzing diseases. We isolated Muse stem cells from patients with an intellectual disability (ID) and mutations in the IQSEC2 gene (i.e., BRAG1 gene) and induced in vitro neuroglial differentiation to study cell commitment and the differentiation of neural lineages. The neuroglial differentiation of Muse cells revealed that IQSEC2 mutations may alter the self-renewal and lineage specification of stem cells. We observed a decrease in the percentage of SOX2 (+) neural stem cells and neural progenitors (i.e., SOX2+ and NESTIN+) in cultures obtained from Muse cells with the mutated IQSEC2 gene. The alteration in the number of stem cells and progenitors produced a bias toward the astrocytes' differentiation. Our research demonstrates that Muse stem cells may represent a new cell-based disease model.

ZFP462 safeguards neural lineage specification by targeting G9A/GLP-mediated heterochromatin to silence enhancers.

ZNF462 haploinsufficiency is linked to Weiss-Kruszka syndrome, a genetic disorder characterized by neurodevelopmental defects, including autism. Though conserved in vertebrates and essential for embryonic development, the molecular functions of ZNF462 remain unclear. We identified its murine homologue ZFP462 in a screen for mediators of epigenetic gene silencing. Here we show that ZFP462 safeguards neural lineage specification of mouse embryonic stem cells (ESCs) by targeting the H3K9-specific histone methyltransferase complex G9A/GLP to silence meso-endodermal genes. ZFP462 binds to transposable elements that are potential enhancers harbouring pluripotency and meso-endoderm transcription factor binding sites. Recruiting G9A/GLP, ZFP462 seeds heterochromatin, restricting transcription factor binding. Loss of ZFP462 in ESCs results in increased chromatin accessibility at target sites and ectopic expression of meso-endodermal genes. Taken together, ZFP462 confers lineage and locus specificity to the broadly expressed epigenetic regulator G9A/GLP. Our results suggest that aberrant activation of lineage non-specific genes in the neuronal lineage underlies ZNF462-associated neurodevelopmental pathology.

Surface Engineering of Auxetic Scaffolds for Neural and Vascular Differentiation from Human Pluripotent Stem Cells.

Auxetic materials are the materials that can display negative Poisson's ratio that describes the degree to which a material contracts (or expands) transversally when axially strained. Human stem cells sense the mechanical properties of the microenvironment, including material surface properties, stiffness, and Poisson's ratio. In this study, six different auxetic polyurethane (PU) foams with different elastic modulus (0.7-1.8kPa) and Poisson's ratio (-0.1 to -0.5) are used to investigate lineage specification of human induced pluripotent stem cells (hiPSCs). The surfaces of the foams are modified with chitosan or heparin to enhance the adhesion and proliferation of hiPSCs. Then, the vascular and neural differentiation of hiPSCs are investigated on different foams with distinct elastic modulus and Poisson's ratio. With different auxetic foams, cells show differential adherent density and differentiation capacity. Chitosan and heparin surface functionalization promote the hindbrain and hippocampal markers, but not forebrain markers during neural patterning of hiPSCs. Properly surface engineered auxetic scaffolds can also promote vascular differentiation of hiPSCs. This study represents a versatile and multifunctional scaffold fabrication approach and can lead to a suitable system for establishing hiPSC culture models in applications of neurovascular disease modeling and drug screening.