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Environmental stiffness is a crucial determinant of cell function. There is a long-standing quest for reproducible and (human matrix) bio-mimicking biomaterials with controllable mechanical properties to unravel the relationship between stiffness and cell behavior. Here, we evaluate methacrylated human recombinant collagen peptide (RCPhC1-MA) hydrogels as a matrix to control 3D microenvironmental stiffness and monitor cardiac cell response. We show that RCPhC1-MA can form hydrogels with reproducible stiffness in the range of human developmental and adult myocardium. Cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (cFBs) remain viable for up to 14 days inside RCPhC1-MA hydrogels while the effect of hydrogel stiffness on extracellular matrix production and hPSC-CM contractility can be monitored in real-time. Interestingly, whereas the beating behavior of the hPSC-CM monocultures is affected by environmental stiffness, this effect ceases when cFBs are present. Together, we demonstrate RCPhC1-MA to be a promising candidate to mimic and control the 3D biomechanical environment of cardiac cells.

Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (Vm), and modulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient Vm hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology.

The histone demethylase KDM1A is a multi- faceted regulator of vital developmental processes, including mesodermal and cardiac tube formation during gastrulation. However, it is unknown whether the fine-tuning of KDM1A splicing isoforms, already shown to regulate neuronal maturation, is crucial for the specification and maintenance of cell identity during cardiogenesis. Here, we discovered a temporal modulation of ubKDM1A and KDM1A+2a during human and mice fetal cardiac development and evaluated their impact on the regulation of cardiac differentiation. We revealed a severely impaired cardiac differentiation in KDM1A(-/-) hESCs that can be rescued by re-expressing ubKDM1A or catalytically impaired ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. Conversely, KDM1A+2a(-/-) hESCs give rise to functional cardiac cells, displaying increased beating amplitude and frequency and enhanced expression of critical cardiogenic markers. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants, independent of their enzymatic activity, during hESC differentiation into cardiac cells.

Publisher Copyright: © 2022 The Author(s) MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with β3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-β receptor II (TGFBR2), a key modulator of TGF-β signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.

Retinal dystrophies (RDs) lead to irreversible vision impairment with no radical treatment. Although photoreceptor cells (PRCs) differentiated from human induced pluripotent stem cells (iPSCs) are essential for the study of RDs as a scalable source, current differentiation methods for PRCs require multiple steps. To address these issues, we developed a method to generate PRCs from human iPSCs by introducing the transcription factors, CRX and NEUROD1. This approach enabled us to generate induced photoreceptor-like cells (iPRCs) expressing PRC markers. Single-cell RNA sequencing revealed the transcriptome of iPRCs in which the genes associated with phototransduction were expressed. Generated iPRCs exhibited their functional properties in calcium imaging. Furthermore, light-induced damage on iPRCs was inhibited by an antioxidant compound. This simple approach would facilitate the availability of materials for PRC-related research and provide a useful application for disease modeling and drug discovery., ヒトiPS細胞からの視細胞直接誘導法 --視細胞を迅速かつ簡便に分化誘導することが可能に--. 京都大学プレスリリース. 2022-03-29.

The Mammary gland undergoes complicated epithelial remodeling to form lobuloalveoli during pregnancy, in which basal epithelial cells remarkably increase to form a basket-like architecture. However, it remains largely unknown how dormant mammary basal stem/progenitor cells involve in lobuloalveolar development. Here, we show that Nfatc1 expression marks a rare population of mammary epithelial cells with the majority being basal epithelial cells. Nfatc1 reporter-marked basal epithelial cells are relatively dormant mammary stem/progenitor cells. Although Nfatc1 reporter-marked basal epithelial cells have limited contribution to the homeostasis of mammary epithelium, they divide rapidly during pregnancy and contribute to lobuloalveolar development. Furthermore, Nfatc1 reporter-marked basal epithelial cells are preferentially used for multiple pregnancies. Using single-cell RNA-seq analysis, we identify multiple functionally distinct clusters within the Nfatc1 reporter-marked cell-derived progeny cells during pregnancy. Taken together, our findings underscore Nfatc1 reporter-marked basal cells as dormant stem/progenitor cells that contribute to mammary lobuloalveolar development during pregnancy.

Summary The Wnt/β-catenin pathway is involved in development, cancer, and embryonic stem cell (ESC) maintenance; its dual role in stem cell self-renewal and differentiation is still controversial. Here, by applying an in vitro system enabling inducible gene expression control, we report that moderate induction of transcriptionally active exogenous β-catenin in β-catenin null mouse ESCs promotes epiblast-like cell (EpiLC) derivation in vitro. Instead, in wild-type cells, moderate chemical pre-activation of the Wnt/β-catenin pathway promotes EpiLC in vitro derivation. Finally, we suggest that moderate β-catenin levels in β-catenin null mouse ESCs favor early stem cell commitment toward mesoderm if the exogenous protein is induced only in the “ground state” of pluripotency condition, or endoderm if the induction is maintained during the differentiation. Overall, our results confirm previous findings about the role of β-catenin in pluripotency and differentiation, while indicating a role for its doses in promoting specific differentiation programs., Graphical abstract, Highlights • Moderate β-catenin levels promote EpiLCs derivation in vitro • Chemical pre-activation of the Wnt pathway enhances ESC-EpiLC transition • β-catenin overexpression tips the balance between mesoderm and endoderm • Cell fate is influenced by the extent of β-catenin induction, Cell biology; Stem cells research

SUMMARYLess than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats for the species and lead to a continuous population decline. To stop erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes, and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes.Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting rescue of this critically endangered species.HIGHLIGHTS-Characterization of Sumatran Rhino (SR) fibroblasts-Generation of SR induced pluripotent stem cells (SR-iPSCs)-SR-iPSCs generate cells of the three germ layers-SR-iPSCs give rise to cerebral organoids

DNA-damage response of cutaneous interfollicular melanocytes to fractionated radiotherapy was investigated by immunostaining of tissue sections from punch biopsies collected before, during, and after the treatment of patients for breast cancer. Our clinical assay with sterilized hair follicles, excluded the migration of immature melanocytes from the bulge, and highlighted interfollicular melanocytes as an autonomous self-renewing population. About thirty percent are immature. Surrounding keratinocytes induced and maintained melanocyte differentiation as long as treatment was ongoing. Concomitant with differentiation, melanocytes were protected from apoptosis by transient upregulation of Bcl-2 and CXCR2. CXCR2 upregulation also indicated the instigation of premature senescence, preventing proliferation. The stem cell factor BMI1 was constitutively expressed exclusively in interfollicular melanocytes and further upregulated upon irradiation. BMI1 prevents apoptosis, terminal differentiation, and premature senescence, allowing dedifferentiation post-treatment, by suppressing the p53/p21-and p16-mediated response and upregulating CXCR2 to genotoxic damage. The pre-treatment immature subset of interfollicular melanocytes was restored after the exposure ended.

Summary Tau pathobiology has emerged as a key component underlying Alzheimer's disease (AD) progression; however, human neuronal in vitro models have struggled to recapitulate tau phenomena observed in vivo. Here, we aimed to define the minimal requirements to achieve endogenous tau aggregation in functional neurons utilizing human induced pluripotent stem cell (hiPSC) technology. Optimized hiPSC-derived cortical neurons seeded with AD brain-derived competent tau species or recombinant tau fibrils displayed increases in insoluble, endogenous tau aggregates. Importantly, MAPT-wild type and MAPT-mutant hiPSC-neurons exhibited unique propensities for aggregation dependent on the seed strain rather than the repeat domain identity, suggesting that successful templating of the recipient tau may be driven by the unique conformation of the seed. The in vitro model presented here represents the first successful demonstration of combining human neurons, endogenous tau expression, and AD brain-derived competent tau species, offering a more physiologically relevant platform to study tau pathobiology., Graphical abstract, Highlights • Seeded tau aggregation in hiPSC-neurons does not require tau overexpression • Tau aggregation is concentration, time, and maturation dependent • Successful templating requires compatibility between neuronal tau and added seeds • Endogenous tau aggregates exhibit properties consistent with pathological tau, Molecular neuroscience; Cellular neuroscience; Stem cells research

Summary Cancer cell stemness (CCS) plays critical roles in both malignancy maintenance and metastasis, yet the underlying molecular mechanisms are far from complete. Although the importance of SOX2 in cancer development and CCS are well recognized, the role of MTA3 in these processes is unknown. In this study, we used esophageal squamous cell carcinoma (ESCC) as a model system to demonstrate that MTA3 can repress both CCS and metastasis in vitro and in vivo. Mechanistically, by forming a repressive complex with GATA3, MTA3 downregulates SOX2OT, subsequently suppresses the SOX2OT/SOX2 axis, and ultimately represses CCS and metastasis. More importantly, MTA3low/SOX2high is associated with poor prognosis and could serve as an independent prognostic factor. These findings altogether indicate that MTA3/SOX2OT/SOX2 axis plays an indispensable role in CCS. Therefore, this axis could be potentially used in cancer stratification and serves as a therapeutic target., Graphical Abstract, Highlights • MTA3 is recruited by GATA3 to repress SOX2OT transcription • The MTA3/GATA3 complex dampens SOX2 signaling by inhibiting SOX2OT transcription • MTA3 can repress both cancer stemness and metastasis of squamous esophageal cancer • Low MTA3 and high SOX2 expression predicts poor prognosis in esophageal cancer, Biological Sciences; Molecular Biology; Cell Biology; Stem Cells Research; Cancer

Summary Epithelia of the oral cavity exhibit variations in morphologies and turnover rates. Are these differences related to environment or to region-specific stem cell populations? A lineage-tracing strategy allowed visualization of Wnt-responsive cells, and their progeny, in the hard and soft palates. In both anatomic locations, Wnt-responsive basal cells self-renewed and gave rise to supra-basal cells. Palatal injuries triggered an enlargement of this population, and their descendants were responsible for wound re-epithelialization. Compared with the hard palate, soft palate stem cells exhibited an earlier, more robust burst in proliferation, culminating in significantly faster repair. Thereafter, excess Wnt-responsive basal cells were removed, and stem cell numbers were restored back to homeostatic level. Thus, we uncovered a stem cell population in oral mucosa, and its relative abundance is correlate with the rate of oral wound healing. Besides the activation during injury, an endogenous mechanism exists to constrain the stem cell pool after repair., Graphical Abstract, Highlights • During homeostasis, long-lived stem cells in the oral epithelium are Wnt responsive • After injury, the soft palate re-epithelizes faster than the hard palate • Faster healing is attributable to an abundance of Wnt-responsive cells • After healing, the Wnt-responsive stem cell pool returns to homeostatic levels, Biological Sciences; Cell Biology; Stem Cells Research

Summary 2-Methylthio-N6-isopentenyl modification of adenosine (ms2i6A) is an evolutionally conserved modification found in mitochondrial (mt)-tRNAs. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) specifically converts N6-isopentenyladenosine (i6A) to ms2i6A at position A37 of four mt-DNA-encoded tRNAs, and the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Here, we report that the ms2 conversion mediated by CDK5RAP1 in mt-tRNAs is required to sustain glioma-initiating cell (GIC)-related traits. CDK5RAP1 maintained the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs. This regulation was not related to the translational control of mt-proteins. CDK5RAP1 abrogated the antitumor effect of i6A by converting i6A to ms2i6A and protected GICs from excessive autophagy triggered by i6A. The elevated activity of CDK5RAP1 contributed to the amelioration of the tumor-suppressive effect of i6A and promoted GIC maintenance. This work demonstrates that CDK5RAP1 is crucial for the detoxification of endogenous i6A and that GICs readily utilize this mechanism for survival., Graphical Abstract, Highlights • CDK5RAP1 is required to sustain the growth of GICs through ms2 modification of i6A • Deficit of CDK5RAP1 inhibits the growth of GIC through i6A accumulation • CDK5RAP1 detoxifies i6A by conversion into ms2i6A in the mitochondria of GICs • Mitochondria serve as antidotal machinery against i6A in GICs, Biological Sciences; Molecular Biology; Cell Biology; Stem Cells Res earch; Cancer

Summary Pancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming., Graphical Abstract, Highlights • Genome-wide identification of miRNAs regulated in human endocrine cell development • Endocrine-enriched miRNAs promote cell cycle exit and endocrine cell formation • Network modeling predicts miRNA-regulated TFs and downstream cell cycle regulators, Molecular Mechanism of Gene Regulation; Molecular Network; Endocrinology; Stem Cells Research

Summary Cells with mesenchymal stem cell properties have been identified in menstrual blood and termed menstrual blood-derived stem/stromal cells (MenSCs). MenSCs have been proposed as ideal candidates for cell-based therapy in regenerative medicine and immune-related diseases. However, MenSCs identity has been loosely defined so far and there is controversy regarding their cell markers and differentiation potential. In this review, we outline the origin of MenSCs in the context of regenerating human endometrium, with attention to endometrial eMSCs as reference cells to understand MenSCs. We summarize the cell identity markers analyzed and the immunomodulatory and reparative properties reported. We also address the recent use of MenSCs in cell reprogramming. The main goal of this review is to contribute to the understanding of the identity and properties of MenSCs as well as to identify potential caveats and new venues that deserve to be explored to strengthen their potential applications., Graphical abstract, Reproductive medicine; Cell biology; Stem cells research

Summary Deciphering the regulatory network for human naive and primed pluripotency is of fundamental theoretical and applicable significance. Here, by combining quantitative proteomics, phosphoproteomics, and acetylproteomics analyses, we revealed RNA processing and translation as the most differentially regulated processes between naive and primed human embryonic stem cells (hESCs). Although glycolytic primed hESCs rely predominantly on the eukaryotic initiation factor 4E (eIF4E)-mediated cap-dependent pathway for protein translation, naive hESCs with reduced mammalian target of rapamycin complex (mTORC1) activity are more tolerant to eIF4E inhibition, and their bivalent metabolism allows for translating selective mRNAs via both eIF4E-dependent and eIF4E-independent/eIF4A2-dependent pathways to form a more compact naive proteome. Globally up-regulated proteostasis and down-regulated post-translational modifications help to further refine the naive proteome that is compatible with the more rapid cycling of naive hESCs, where CDK1 plays an indispensable coordinative role. These findings may assist in better understanding the unrestricted lineage potential of naive hESCs and in further optimizing conditions for future clinical applications, Graphical Abstract, Highlights • RNA processing and translation are most different between naive and primed hESCs • Glycolytic primed hESCs mainly rely on eIF4E-dependent translation • Bivalent metabolism in naive hESCs promotes eIF4E-independent translation • CDK1 is required for naive pluripotency partially by activating E-cadherin signaling, Molecular biology; Stem cells research; Proteomics

Summary We have reconciled steady-state and stress hematopoiesis in a single mathematical model based on murine in vivo experiments and with a focus on hematopoietic stem and progenitor cells. A phenylhydrazine stress was first applied to mice. A reduced cell number in each progenitor compartment was evidenced during the next 7 days through a drastic level of differentiation without proliferation, followed by a huge proliferative response in all compartments including long-term hematopoietic stem cells, before a return to normal levels. Data analysis led to the addition to the 6-compartment model, of time-dependent regulation that depended indirectly on the compartment sizes. The resulting model was finely calibrated using a stochastic optimization algorithm and could reproduce biological data in silico when applied to different stress conditions (bleeding, chemotherapy, HSC depletion). In conclusion, our multi-step and time-dependent model of immature hematopoiesis provides new avenues to a better understanding of both normal and pathological hematopoiesis., Graphical abstract, Highlights • We describe a new 6-compartment time-dependent regulated model of hematopoiesis • Biological data under steady state and stress and cell dynamics were used • Modeling is able to recapitulate effects from chemotherapy, bleeding, or HSC depletion, Cell biology; Stem cells research; In silico biology

Summary Nuclear transfer systems represent the efficient means to reprogram a cell and in theory provide a basis for investigating the development of endangered species. However, conventional nuclear transfer using oocytes of laboratory animals does not allow reprogramming of cross-species nuclei owing to defects in cell divisions and activation of embryonic genes. Here, we show that somatic nuclei transferred into mouse four-cell embryos arrested at the G2/M phase undergo reprogramming toward the embryonic state. Remarkably, genome-wide transcriptional reprogramming is induced within a day, and ZFP281 is important for this replication-free reprogramming. This system further enables transcriptional reprogramming of cells from Oryx dammah, now extinct in the wild. Thus, our findings indicate that arrested mouse embryos are competent to induce intra- and cross-species reprogramming. The direct induction of embryonic transcripts from diverse genomes paves a unique approach for identifying mechanisms of transcriptional reprogramming and genome activation from a diverse range of species., Graphical abstract, Highlights • Mouse embryos arrested at the four-cell stage can induce transcriptional reprogramming • ZFP281 plays a role in transcriptional reprogramming • Embryonic environment permits cell division- and DNA replication-free reprogramming • Transcriptional reprogramming of cross-species nuclei is induced in mouse embryos, Cell biology; Stem cells research; Developmental biology

Human pluripotent stem cells (hPSCs) are an exciting and promising source to enable cell replacement therapies for a variety of unmet medical needs. Though hPSCs can be successfully derived into numerous physiologically relevant cell types, effective translation to the clinic is limited by challenges in scalable production of high-quality cells, cellular immaturity following the differentiation process, and the use of animal-derived components in culture. To address these limitations, we have developed a fully defined, reproducible, and tunable thermoreversible polymer for high-quality, scalable 3D cell production. Our reproducible synthesis method enables precise control of gelation temperature (24°C-32°C), hydrogel stiffness (100-4000 Pa), and the prevention of any unintended covalent crosslinking. After material optimization, we demonstrated hPSC expansion, pluripotency maintenance, and differentiation into numerous lineages within the hydrogel. Overall, this 3D thermoreversible hydrogel platform has broad applications in scalable, high-quality cell production to overcome the biomanufacturing burden of stem cell therapy.

Summary: Because epigenetics is a critical component for gene expression, the hypothesis was tested that DNA methylation alterations are dynamic and continually change throughout gametogenesis to generate the mature sperm. Developmental alterations and stage-specific DNA methylation during gametogenesis from primordial germ cells (PGCs) to mature sperm are investigated. Individual developmental stage germ cells were isolated and analyzed for differential DNA methylation regions (DMRs). The number of DMRs was highest in the first three comparisons with mature PGCs, prospermatogonia, and spermatogonia. The most statistically significant DMRs were present at all stages of development and had variations involving both increases or decreases in DNA methylation. DMR-associated genes were identified and correlated with gene functional categories, pathways, and cellular processes. Observations identified a dynamic cascade of epigenetic changes during development that is dramatic during the early developmental stages. Complex epigenetic alterations are required to regulate genome biology and gene expression during gametogenesis.

Summary Although cell density is known to affect numerous biological processes including gene expression and cell fate specification, mechanistic understanding of what factors link cell density to global gene regulation is lacking. Here, we reveal that the expression of thousands of genes in mouse embryonic stem cells (mESCs) is affected by cell seeding density and that low cell density enhances the efficiency of differentiation. Mechanistically, β-catenin is localized primarily to adherens junctions during both self-renewal and differentiation at high density. However, when mESCs differentiate at low density, β-catenin translocates to the nucleus and associates with Tcf7l1, inducing co-occupied lineage markers. Meanwhile, Esrrb sustains the expression of pluripotency-associated genes while repressing lineage markers at high density, and its association with DNA decreases at low density. Our results provide new insights into the previously neglected but pervasive phenomenon of density-dependent gene regulation., Graphical abstract, Highlights • Cell density influences expression of thousands of genes in embryonic stem cells • Low seeding density augments differentiation efficiency via β-catenin • β-catenin works in tandem with Tcf7l1 for lineage marker induction • Esrrb opposes the action of β-catenin and Tcf7l1 by repressing their targets, Cell biology; Stem cells research; Transcriptomics

Summary Skeletal muscle is composed of post-mitotic myofibers that form a syncytium containing hundreds of myonuclei. Using a progressive exercise training model in the mouse and single nucleus RNA-sequencing (snRNA-seq) for high-resolution characterization of myonuclear transcription, we show myonuclear functional specialization in muscle. After 4 weeks of exercise training, snRNA-seq reveals that resident muscle stem cells, or satellite cells, are activated with acute exercise but demonstrate limited lineage progression while contributing to muscle adaptation. In the absence of satellite cells, a portion of nuclei demonstrates divergent transcriptional dynamics associated with mixed-fate identities compared with satellite cell replete muscles. These data provide a compendium of information about how satellite cells influence myonuclear transcription in response to exercise., Graphical abstract, Highlights • Single nucleus RNA-sequencing, trajectory inference, and entropy analyses • Genetic depletion of satellite cells before exercise training in adult mice • Dysregulated nuclei arise 24 h after last exercise bout when without satellite cells • Satellite cell depletion alters myonuclear transcription during growth and adaptation, Biological sciences; Stem cells research; Transcriptomics

Summary Pluripotent embryonic stem cells (ESCs) constitute the cell types of the adult vertebrate through a series of developmental state transitions. These states can be defined by expression levels of marker genes, such as Nanog and Sox2. In culture, ESCs reversibly transition between states. However, whether ESCs retain memory of their previous states or transition in a memoryless (Markovian) process remains relatively unknown. Here, we show some highly dynamic lineages of ESCs do not exhibit the Markovian property: their previous states and kin relations influence future choices. Unexpectedly, the distribution of lineages across their composition between states is constant over time, contrasting with the predictions of a Markov model. Additionally, highly dynamic ESC lineages show skewed cell fate distributions after retinoic acid differentiation. Together, these data suggest ESC lineage is an important variable governing future cell states, with implications for stem cell function and development., Graphical abstract, Highlights • Dynamics of embryonic stem cells switching between Nanog and Sox2 defined states • Lineage-level dynamics are non-Markov over a time period of days to weeks • A subset of lineages shows high transition rate between states • These lineages skew cell fate under retinoic acid differentiation, Cell biology; Stem cells research; Developmental biology; Embryology; Systems and computational biology

Summary: Metabolism is vital to cellular function and tissue homeostasis during human lung development. In utero, embryonic pluripotent stem cells undergo endodermal differentiation toward a lung progenitor cell fate that can be mimicked in vitro using induced human pluripotent stem cells (hiPSCs) to study genetic mutations. To identify differences between wild-type and surfactant protein B (SFTPB)-deficient cell lines during endoderm specification toward lung, we used an untargeted metabolomics approach to evaluate the developmental changes in metabolites. We found that the metabolites most enriched during the differentiation from pluripotent stem cell to lung progenitor cell, regardless of cell line, were sphingomyelins and phosphatidylcholines, two important lipid classes in lung development. The SFTPB mutation had no metabolic impact on early endodermal lung development. The identified metabolite signatures during lung progenitor cell differentiation may be utilized as biomarkers for normal embryonic lung development.

Summary Prostaglandin E2 (PGE2) has recently been recognized to play a role in immune regulation and tissue regeneration. However, the short half-life of PGE2 limits its clinical application. Improving the delivery of PGE2 specifically to the target organ with a prolonged release method is highly desirable. Taking advantage of the adequate space and proximity of the renal parenchyma, renal subcapsular delivery allows minimally invasive and effective delivery to the entire kidney. Here, we report that by covalently cross-linking it to a collagen matrix, PGE2 exhibits an adequate long-term presence in the kidney with extensive intraparenchymal penetration through renal subcapsular delivery and significantly improves kidney function. Sox9 cell lineage tracing with intravital microscopy revealed that PGE2 could activate the endogenous renal progenitor Sox9+ cells through the Yap signaling pathway. Our results highlight the prospects of utilizing renal subcapsular-based drug delivery and facilitate new applications of PGE2-releasing matrices for regenerative therapy., Graphical abstract, Highlights • PGE2 exhibits an adequate long-term release by being covalently cross-linked to collagen • The renal subcapsular space serves as a reservoir for the delivery of PGE2 • Sox9+ renal progenitor cells can be lineage traced intravitally by microscopy • PGE2 activates the endogenous renal progenitor Sox9+ cells through the YAP pathway, Drug delivery system; Pathophysiology; Cell biology; Stem cells research

Summary Human induced pluripotent stem cells (hiPSCs) can generate a multiplicity of organoids, yet no compelling evidence currently exists as to whether or not these contain tissue-specific, holoclone-forming stem cells. Here, we show that a subpopulation of cells in a hiPSC-derived corneal epithelial cell sheet is positive for ABCB5 (ATP-binding cassette, sub-family B, member 5), a functional marker of adult corneal epithelial stem cells. These cells possess remarkable holoclone-forming capabilities, which can be suppressed by an antibody-mediated ABCB5 blockade. The cell sheets are generated from ABCB5+ hiPSCs that first emerge in 2D eye-like organoids around six weeks of differentiation and display corneal epithelial immunostaining characteristics and gene expression patterns, including sustained expression of ABCB5. The findings highlight the translational potential of ABCB5-enriched, hiPSC-derived corneal epithelial cell sheets to recover vision in stem cell-deficient human eyes and represent the first report of holoclone-forming stem cells being directly identified in an hiPSC-derived organoid., Graphical abstract, Highlights • Human iPS cell-derived corneal epithelia contain ABCB5-positive stem cells • The ABCB5-positive cells possess holoclone-forming capabilities • An antibody-mediated ABCB5 blockade suppresses holoclone formation • Holoclone-forming stem cells are present in a human iPS cell-derived tissue construct, Bioengineering; Cell biology; Stem cells research; Tissue engineering

Summary Despite increasing knowledge on primed and naive pluripotency, the cell signaling that regulates the pluripotency type in stem cells remains not fully understood. Here we show that AMP kinase (AMPK) activators can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to the naive pluripotent state. The addition of AMPK activators alone or together with leukemia inhibitory factor to primed mEpiSCs induced the appearance of naive-like cells. After passaging in naive culture conditions, the colony morphology, protein expression, and global gene expression profiles indicated the naive state, as did germline transmission ability. Loss-of-function and gain-of-function studies suggested that p38 is a critical downstream target in AMPK activation. Finally, single-cell RNA sequencing analysis revealed that the reversion process through AMPK signaling passes an intermediate naive-like population. In conclusion, the AMPK pathway is a critical driving force in the reversion of primed to naive pluripotency., Graphical abstract, Highlights • AMPK activation can induce mouse primed stem cell reversion to naive state • Reverted cells fully obtain naive pluripotency • p38 is a critical downstream molecule of AMPK pathway in the reversion process • Dppa5 and Dazl are highly expressed in intermediate reversion cells, Cell biology; Stem cells research; Transcriptomics

Summary Wnt/β-catenin signaling is regulated in a bimodal fashion during cardiogenesis. Signaling is initially required to promote generation of precardiac mesoderm, but subsequently must be repressed for cardiac progenitor specification. TMEM88 was discovered recently as a negative regulator during the later phase of cardiac progenitor specification, but how TMEM88 functions was unknown. Based on a C-terminal PDZ-binding motif, TMEM88 was proposed to act by targeting the PDZ domain of Dishevelled, the positive Wnt signaling mediator. However, we discovered that TMEM88 acts downstream of the β-catenin destruction complex and can inhibit Wnt signaling independent of Dishevelled. TMEM88 requires the PDZ-binding motif for trafficking from Golgi to the plasma membrane and is also found in the multivesicular body (MVB) associated with the endocytosed Wnt signalosome. Expression of Tmem88 promotes association of the Wnt signalosome including β-catenin to the MVB, leading to reduced accumulation of nuclear β-catenin and repression of Wnt signaling., Graphical Abstract, Highlights • Human ESCs with a targeted TMEM88 knockout are impaired for cardiac specification • TMEM88 does not require Dishevelled to inhibit Wnt signaling • TMEM88 is trafficked from Golgi to plasma membrane and then to the MVB • Expression of TMEM88 promotes association of the signalosome to the MVB, Molecular Mechanism of Behavior; Cell Biology; Stem Cells Research

Summary The generation of humanized ectopic ossicles (hOss) in mice has been proposed as an advanced translational and fundamental model to study the human hematopoietic system. The approach relies on the presence of human bone marrow-derived mesenchymal stromal cells (hMSCs) supporting the engraftment of transplanted human hematopoietic stem and progenitor cells (HSPCs). However, the functional distribution of hMSCs within the humanized microenvironment remains to be investigated. Here, we combined genetic tools and quantitative confocal microscopy to engineer and subsequently analyze hMSCs′ fate and distribution in hOss. Implanted hMSCs reconstituted a humanized environment including osteocytes, osteoblasts, adipocytes, and stromal cells associated with vessels. By imaging full hOss, we identified rare physical interactions between hMSCs and human CD45+/CD34+/CD90+ cells, supporting a functional contact-triggered regulatory role of hMSCs. Our study highlights the importance of compiling quantitative information from humanized organs, to decode the interactions between the hematopoietic and the stromal compartments., Graphical Abstract, Highlights • Mesenchymal cells can generate human bone organs with tailored molecular signature • Mesenchymal cells reconstitute a human niche environment capable of regulating HSPCs, Biological Sciences; Stem Cells Research; Tissue Engineering

Summary Bacteria use flagella, the protein nanofibers on their surface, as a molecular machine to swim. Flagella are polymerized from monomers, flagellins, which can display a peptide by genetic means. However, flagella as genetically modifiable nanofibers have not been used in building bone extracellular matrix-like structures for inducing stem cell differentiation in non-osteogenic medium. Here we discovered that interactions between Ca2+ ions and flagella (displaying a collagen-like peptide (GPP)8 on every flagellin) resulted in ordered bundle-like structures, which were further mineralized with hydroxyapatite to form ordered fibrous matrix. The resultant matrix significantly induced the osteogenic differentiation of stem cells, much more efficiently than wild-type flagella and type I collagen. This work shows that flagella can be used as protein building blocks in generating biomimetic materials., Graphical Abstract, Highlights • Flagella as genetically modifiable protein nanofibers for bacterial swimming • Genetically engineering the flagella to surface display collagen-like peptides • Assembly and mineralization of flagella into mineralized collagen-like structures • Osteogenic differentiation of stem cells induced by mineralized collagen-like matrix, Genetic Engineering; Stem Cells Research; Materials Science; Biomaterials

Summary During mammalian development, liver differentiation is driven by signals that converge on multiple transcription factor networks. The hepatocyte nuclear factor signaling network is known to be essential for hepatocyte specification and maintenance. In this study, we have generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4α) to precisely evaluate the function of protein domains during hepatocyte specification from human pluripotent stem cells. We demonstrate that nuclear HNF4α is essential for hepatic progenitor specification, and the introduction of point mutations in HNF4α′s Small Ubiquitin-like Modifier (SUMO) consensus motif leads to disrupted hepatocyte differentiation. Taking a multiomics approach, we identified key deficiencies in cell biology, which included dysfunctional metabolism, substrate adhesion, tricarboxylic acid cycle flux, microRNA transport, and mRNA processing. In summary, the combination of genome editing and multiomics analyses has provided valuable insight into the diverse functions of HNF4α during pluripotent stem cell entry into the hepatic lineage and during hepatocellular differentiation., Graphical Abstract, Highlights • Deletion of HNF4α's DNA-binding domain blocks hepatic progenitor formation • HNF4α′s C-terminal SUMO consensus motif plays roles in hepatocyte specification • Multiomics analyses reveal various processes regulated by HNF4α during differentiation, Biological Sciences; Cell Biology; Developmental Biology; Stem Cells Research

Summary A low surface expression level of human leukocyte antigen class I (HLA-I) ensures that the mesenchymal stem cells (MSCs) escape from the allogeneic recipients' immunological surveillance. Here, we discovered that both transcriptional and synthesis levels of HLA-I in MSCs increased continuously after interferon (IFN)-γ treatment, whereas interestingly, their surface HLA-I expression was downregulated after reaching an HLA-I surface expression peak. Microarray data indicated that the post-transcriptional process plays an important role in the downregulation of surface HLA-I. Further studies identified that IFN-γ-treated MSCs accelerated HLA-I endocytosis through a clathrin-independent dynamin-dependent endocytosis pathway. Furthermore, cells that have self-downregulated surface HLA-I expression elicit a weaker immune response than they previously could. Thus uncovering the plasticity of MSCs in the regulation of HLA-I surface expression would reveal insights into the membrane transportation events leading to the maintenance of low surface HLA-I expression, providing more evidence for selecting and optimizing low-immunogenic MSCs to improve the therapeutic efficiency., Graphical Abstract, Highlights • hESC-MSCs have the plasticity of maintaining low HLA-I expression on cell surface • hESC-MSCs downregulate the surface HLA-I expression through endocytosis of HLA-I • hESC-MSCs with lower HLA-I surface expression induce weaker MLR and slighter DTH, Biological Sciences; Immunology; Cell Biology; Stem Cells Research

Summary Human pluripotent stem cells (hPSCs) acquire genetic changes during their propagation in culture that can affect their use in research and future therapies. To identify the key genes involved in selective advantage during culture adaptation and tumorigenicity of hPSCs, we generated a genome-wide screening system for genes and pathways that provide a growth advantage either in vitro or in vivo. We found that hyperactivation of the RAS pathway confers resistance to selection with the hPSC-specific drug PluriSIn-1. We also identified that inactivation of the RHO-ROCK pathway gives growth advantage during culture adaptation. Last, we demonstrated the importance of the PI3K-AKT and HIPPO pathways for the teratoma formation process. Our screen revealed key genes and pathways relevant to the tumorigenicity and survival of hPSCs and should thus assist in understanding and confronting their tumorigenic potential., Graphical Abstract, Highlights • Large-scale analysis of genes and pathways involved in growth and survival of hPSCs • Activation of the RAS pathways confers enhanced resistance to PluriSIn-1 treatment • Inactivation of the RHO-ROCK pathway gives selective growth advantage to hPSCs • The PI3K-AKT and HIPPO pathways are involved in the process of teratoma formation, Cell Biology; Stem Cells Research; Omics; Genomics

Summary Owing to their single genome, haploid cells are powerful to uncover unknown genes by performing genetic screening in mammals. However, no haploid cell line from an extraembryonic lineage has been achieved yet, which limits the application of haploid cells in placental genetic screening. Here, we show that overexpression of Cdx2 can convert haploid embryonic stem cells to trophoblast stem cells (TSCs). p53 deletion reduces diploidization during the conversion and guarantees the generation of haploid-induced TSCs (haiTSCs). haiTSCs not only share the same molecular characterization with trophoderm-derived TSCs but also possess multipotency to placental lineages in various procedures. In addition, haiTSCs can maintain haploidy in the long term, assisted by periodic sorting and with reliance on FGF4 and heparin. Finally, we perform piggyBac-mediated high-throughput mutation in haiTSCs and use them in trophoblast lineage genetic screening. Deep sequencing analysis and validation experiments prove that Htra1 is a blocker for spongiotrophoblast specification., Graphical Abstract, Highlights • A haploid cell line of extraembryonic lineages with self-renewal ability • haiTSCs have multipotency to functional trophoblast lineages both in vitro and in vivo • High-throughput screening of spongiotrophoblast specification-related genes in haiTSCs • Htra1 is a blocker for spongiotrophoblast-specific differentiation, Cell Biology; Stem Cells Research; Genomics

Summary Epidermal homeostasis requires balanced and coordinated adult stem cell renewal and differentiation. These processes are controlled by both extracellular signaling and by cell intrinsic transcription regulatory networks, yet how these control mechanisms are integrated to achieve this is unclear. Here, we developed single-cell Immuno-Detection by sequencing (scID-seq) and simultaneously measured 69 proteins (including 34 phosphorylated epitopes) at single-cell resolution to study the activation state of signaling pathways during human epidermal differentiation. Computational pseudo-timing inference revealed dynamic activation of the JAK-STAT, WNT, and BMP pathways along the epidermal differentiation trajectory. We found that during differentiation, cells start producing BMP2-ligands and activate the canonical intracellular effectors SMAD1/5/9. Mechanistically, the BMP pathway is responsible for activating the MAF/MAFB/ZNF750 transcription factor network to drive late-stage epidermal differentiation. Our work indicates that incorporating signaling pathway activation into this transcription regulatory network enables coordination of transcription programs during epidermal differentiation., Graphical Abstract, Highlights • scID-seq allows quantification of 70 (phospho-)proteins at single-cell level • Pseudo-time inference reveals signaling dynamics during epidermal differentiation • BMP signaling drives a late differentiation transcription program • BMP signaling activates the MAF/MAFB/ZNF750 transcription factor network, Stem Cells Research; Systems Biology; Proteomics

Summary It is still a challenge for synthesizing ‘cellular niche-mimics’ in vitro with satisfactory reproducibility and fidelity to recreate the natural niche components (e.g., extracellular matrices and soluble factors) for stem cell cultivation. Inspired by the massive amplification of hepatic progenitor cells during liver fibrosis in vivo, here we optimized the in vitro liver fibrotic niches and subsequently harvested their bioactive ingredients as niche extracts (NEs). The fibrosis-relevant NE marginally outperformed Matrigel for phenotype maintenance of human embryonic stem cell (hESC)-derived hepatoblasts (HBs) and recapitulation of the pathological angiogenesis of hESC-derived endothelial cells both in 2D culture and 3D liver organoids. Finally, defined NE components (i.e., collagen III, IV, IL-17, IL-18 and M-CSF) were resolved by the quantitative proteomics which exhibited advantage over Matrigel for multi-passaged HB expansion. The pathology-relevant and tissue-specific NEs provide innovative and generalizable strategies for the discovery of optimal cellular niche and bioactive niche compositions., Graphical abstract, Highlights • Fibrotic niches were constructed by 3 hepatic cell lines plus 4 profibrotic factors • NE was produced by enzymatic digestion using pepsin and DNase • Collagen III, IV, IL-17, IL-18, and M-CSF resolved from NE promoted HBs expansion, Tissue engineering; Biomimetics; Stem cells research

Summary Cell therapy using human-stem-cell-derived pancreatic beta cells (hSC-βs) is a potential treatment method for type 1 diabetes mellitus (T1D). For therapeutic safety, hSC-βs need encapsulation in grafts that are scalable and retrievable. In this study, we developed a lotus-root-shaped cell-encapsulated construct (LENCON) as a graft that can be retrieved after long-term hSC-β transplantation. This graft had six multicores encapsulating hSC-βs located within 1 mm from the edge. It controlled the recipient blood glucose levels for a long-term, following transplantation in immunodeficient diabetic mice. LENCON xenotransplanted into immunocompetent mice exhibited retrievability and maintained the functionality of hSC-βs for over 1 year after transplantation. We believe that LENCON can contribute to the treatment of T1D through long-term transplantation of hSC-βs and in many other forms of cell therapy.

Summary Individual cell environment stimulating single cell is a suitable strategy for the generation of sophisticated multicellular aggregates with localized biochemical signaling. However, such strategy for induced pluripotent stem cell (iPSC)-derived embryoid bodies (EBs) is limited because the presence of external stimulation can inhibit spontaneous cellular communication, resulting in misdirection in the maturation and differentiation of EBs. In this study, a facile method of engineering the iPSC membrane to stimulate the inner cell of EBs while maintaining cellular activities is reported. We coated the iPSC surface with nanoscale extracellular matrix fabricated by self-assembly between vitronectin and heparin. This nano-coating allowed iPSC to retain its in vitro properties including adhesion capability, proliferation, and pluripotency during its aggregation. More importantly, the nano-coating did not induce lineage-specific differentiation but increased E-cadherin expression, resulting in promotion of development of EB. This study provides a foundation for future production of sophisticated patient-specific multicellular aggregates by modification of living cell membranes., Graphical abstract, Highlights • VTN/HEP nano-coating acts as a flexible individual cellular environment • VTN/HEP nano-coating stimulates embryoid body to promote its development • VTN/HEP nano-coating preserves spontaneous cell aggregation

Summary Mesenchymal stromal cell-like (MSCl) cells generated from human embryonic stem cells are considered to be an eligible cell line to model the immunomodulatory behavior of mesenchymal stromal cells (MSCs) in vitro. Dendritic cells (DCs) are essential players in the maintenance and restoration of the sensitive balance between tolerance and immunity. Here, the effects of MSCl cells on the in vitro differentiation of human monocytes into DCs were investigated. MSCl cells promote the differentiation of CTLA-4 expressing DCs via the production of all-trans retinoic acid (ATRA) functioning as a ligand of RARα, a key nuclear receptor in DC development. These semi-matured DCs exhibit an ability to activate allogeneic, naive T cells and polarize them into IL-10 + IL-17 + double-positive T helper cells in a CTLA-4-dependent manner. Mapping the molecular mechanisms of MSC-mediated indirect modulation of DC differentiation may help to expand MSCs' clinical application in cell-free therapies., Graphical abstract, Highlights • Mesenchymal stromal cell-like cells alter moDC differentiation via RARα activation • Mesenchymal stromal cell-like cells express genes known to play role in ATRA synthesis • MoDCs, differentiated in the presence of MSCl-derived factors, express CTLA-4 • CTLA-4+ moDCs are able to induce polarization of IL-10- and IL-17-producing helper T cells, Molecular Biology; Immunology; Components of the Immune System; Stem Cells Research

Summary Isolation of long-term hematopoietic stem cell (HSC) is possible by utilizing flow cytometry with multiple cell surface markers. However, those cell surface phenotypes do not represent functional HSCs after in vitro culture. Here we show that cultured HSCs express mast cell-related genes including Cd244. After in vitro culture, phenotypic HSCs were divided into CD244- and CD244+ subpopulations, and only CD244- cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. The result was same when HSCs were cultured in an efficient expansion medium containing polyvinyl alcohol. Chemically induced endoplasmic reticulum (ER) stress signal increased the CD244+ subpopulation, whereas ER stress suppression using a molecular chaperone, TUDCA, decreased CD244+ population, which was correlated to improved reconstitution output. These data suggest CD244 is a potent marker to exclude non-functional HSCs after in vitro culture thereby useful to elucidate mechanism of functional decline of HSCs during ex vivo treatment., Graphical Abstract, Highlights • Murine HSCs up-regulate mast cell-related genes including Cd244 during in vitro culture • Long-term HSCs after in vitro culture are enriched in CD244−CD48−KSL population • Induction of unfolded protein response is involved in the increase of CD244+HSC, Biological sciences; Immunology; Cell biology; Stem cells research

Summary Cancer stem cells, quiescent and drug resistant, have become a therapeutic target. Unlike high-intensity focused ultrasound directly killing tumor, low-intensity pulsed ultrasound (LIPUS), a new noninvasive physical device, promotes pluripotent stem cell differentiation and is primarily applied in tissue engineering but rarely in oncotherapy. We explored the effect and mechanism of LIPUS on glioma stem cell (GSC) expulsion from quiescence. Here, we observed that LIPUS led to attenuated expression of GSC biomarkers, promoted GSC escape from G0 quiescence, and significantly weakened the Wnt and Hh pathways. Of note, LIPUS transferred sonomechanical energy into cytochrome c and B5 proteins, which converted oxygen molecules into singlet oxygen, triggering telomere crisis. The in vivo and in vitro results confirmed that LIPUS enhanced the GSC sensitivity to temozolomide. These results demonstrated that LIPUS “waked up” GSCs to improve their sensitivity to chemotherapy, and importantly, we confirmed the direct targeted proteins of LIPUS in GSCs., Graphical abstract, Highlights • LIPUS “waked quiescent GSCs up” and enhanced the sensitivity of temozolomide • LIPUS-generated singlet oxygen damaged telomeres, reducing GSCs stemness • Cytochrome B5 and c are targets for LIUPS energy transfer, Medical device; Cell biology; Stem cells research; Cancer

Summary: Many acute and chronic diseases affect the distal lung alveoli. Alveolar epithelial cell (AEC) lines are needed to better model these diseases. We used de-identified human remnant transplant lungs to develop a method to establish AEC lines. The lines grow well in 2-dimensional (2D) culture as epithelial monolayers expressing lung progenitor markers. In 3-dimensional (3D) culture with fibroblasts, Matrigel, and specific media conditions, the cells form alveolar-like organoids expressing mature AEC markers including aquaporin 5 (AQP5), G-protein-coupled receptor class C group 5 member A (GPRC5A), and surface marker HTII280. Single-cell RNA sequencing of an AEC line in 2D versus 3D culture revealed increased cellular heterogeneity and induction of cytokine and lipoprotein signaling in 3D organoids. Our approach yields lung progenitor lines that retain the ability to differentiate along the alveolar cell lineage despite long-term expansion and provides a valuable system to model and study the distal lung in vitro.

Summary ESC/iPSC-retinal sheet transplantation, which supplies photoreceptors as well as other retinal cells, has been shown to be able to restore visual function in mice with end-stage retinal degeneration. Here, by introducing a novel type of genetically engineered mouse ESC/iPSC-retinal sheet with reduced numbers of secondary retinal neurons but intact photoreceptor cell layer structure, we reinforced the evidence that ESC/iPSC-retinal sheet transplantation can establish synaptic connections with the host, restore light responsiveness, and reduce aberrant retinal ganglion cell spiking in mice. Furthermore, we show that genetically engineered grafts can substantially improve the outcome of the treatment by improving neural integration. We speculate that this leads to reduced spontaneous activity in the host which in turn contributes to a better visual recovery., Graphical Abstract, Highlights • Photoreceptors in organoids with bipolar cell-KO can functionally mature in vivo • Photoreceptors in KO grafts form more synapses with host bipolar cells • KO grafts better suppress heightened spontaneous spiking in degenerated retina • Mice transplanted with KO grafts perform better in light-guided behavior test, Neuroscience; Cellular neuroscience; Bioengineering; Tissue engineering; Stem cells research

Summary Osteoarthritis (OA) is the most prevalent chronic joint disease that affects a large proportion of the elderly population. Chondrogenic progenitor cells (CPCs) reside in late-stage OA cartilage tissue, producing a fibrocartilaginous extracellular matrix; these cells can be manipulated in vitro to deposit proteins of healthy articular cartilage. CPCs are under the control of SOX9 and RUNX2. In our earlier studies, we showed that a knockdown of RUNX2 enhanced the chondrogenic potential of CPCs. Here we demonstrate that CPCs carrying a knockout of RAB5C, a protein involved in endosomal trafficking, exhibited elevated expression of multiple chondrogenic markers, including the SOX trio, and increased COL2 deposition, whereas no changes in COL1 deposition were observed. We report RAB5C as an attractive target for future therapeutic approaches designed to increase the COL2 content in the diseased joint., Graphical abstract, Highlights • RUNX2 has a pro-chondrogenic effect in CPCs. • RAB5C is a potential interaction partner of SOX9 in CPCs. • Loss of RAB5C increases the chondrogenic potential of CPCs., Stem cells research; Omics; Proteomics ; Transcriptomics

Intestinal metaplasia is related to gastric carcinogenesis. Previous studies have suggested the important role of CDX2 in intestinal metaplasia, and several reports have shown that the overexpression of CDX2 in mouse gastric mucosa caused intestinal metaplasia. However, no study has examined the induction of intestinal metaplasia using human gastric mucosa. In the present study, to produce an intestinal metaplasia model in human gastric mucosa in vitro, we differentiated human-induced pluripotent stem cells (hiPSC) to gastric organoids, followed by the overexpression of CDX2 using a tet-on system. The overexpression of CDX2 induced, although not completely, intestinal phenotypes and the enhanced expression of many, but not all, intestinal genes and previously reported intestinal metaplasia-related genes in the gastric organoids. This model can help clarify the mechanisms underlying intestinal metaplasia and carcinogenesis in human gastric mucosa and develop therapies to restitute precursor conditions of gastric cancer to normal mucosa.

Summary Teeth exert fundamental functions related to mastication and speech. Despite their great biomedical importance, an overall picture of their cellular and molecular composition is still missing. In this study, we have mapped the transcriptional landscape of the various cell populations that compose human teeth at single-cell resolution, and we analyzed in deeper detail their stem cell populations and their microenvironment. Our study identified great cellular heterogeneity in the dental pulp and the periodontium. Unexpectedly, we found that the molecular signatures of the stem cell populations were very similar, while their respective microenvironments strongly diverged. Our findings suggest that the microenvironmental specificity is a potential source for functional differences between highly similar stem cells located in the various tooth compartments and open new perspectives toward cell-based dental therapeutic approaches., Graphical abstract, Highlights • Dental atlas of the pulp and periodontal tissues of human teeth • Identification of three common MSC subclusters between dental pulp and periodontium • Dental pulp and periodontal MSCs are similar, and their niches diverge, Cell Biology; Stem Cells Research; Omics; Transcriptomics

Summary Matrin3 (MATR3) is a nuclear RNA/DNA-binding protein that plays pleiotropic roles in gene expression regulation by directly stabilizing target RNAs and supporting the activity of transcription factors by modulating chromatin architecture. MATR3 is involved in the differentiation of neural cells, and, here, we elucidate its critical functions in regulating pluripotent circuits in human induced pluripotent stem cells (hiPSCs). MATR3 downregulation affects hiPSCs' differentiation potential by altering key pluripotency regulators' expression levels, including OCT4, NANOG, and LIN28A by pleiotropic mechanisms. MATR3 binds to the OCT4 and YTHDF1 promoters favoring their expression. YTHDF1, in turn, binds the m6A-modified OCT4 mRNA. Furthermore, MATR3 is recruited on ribosomes and controls pluripotency regulating the translation of specific transcripts, including NANOG and LIN28A, by direct binding and favoring their stabilization. These results show that MATR3 orchestrates the pluripotency circuitry by regulating the transcription, translational efficiency, and epitranscriptome of specific transcripts., Graphical abstract, Highlights • MATR3 orchestrates the pluripotency circuitry in hiPSCs • MATR3 binds to the OCT4 and YTHDF1 promoters and favors their expression • YTHDF1 binds the m6A-modified OCT4 mRNA • MATR3 regulates the translation of specific transcripts including NANOG and LIN28A, Molecular biology; cellular neuroscience; stem cells research

Summary Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term encompasses a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis, which have, to date, been challenging to model in the laboratory setting. Here, we present a human pluripotent stem cell (hPSC)-derived model of hepatic steatosis, which overcomes inherent challenges of current models and provides insights into the metabolic rewiring associated with steatosis. Following induction of macrovesicular steatosis in hepatocyte-like cells using lactate, pyruvate, and octanoate (LPO), respirometry and transcriptomic analyses revealed compromised electron transport chain activity. 13C isotopic tracing studies revealed enhanced TCA cycle anaplerosis, with concomitant development of a compensatory purine nucleotide cycle shunt leading to excess generation of fumarate. This model of hepatic steatosis is reproducible, scalable, and overcomes the challenges of studying mitochondrial metabolism in currently available models., Graphical Abstract, Highlights • LPO-exposed hPSC-derived hepatocyte-like cells resemble human hepatic steatosis • Steatosis is associated with decreased mitochondrial maximal respiration • Purine nucleotide cycle activity increases in steatotic hepatocytes • Excess fumarate production is associated with induction of macrovesicular steatosis, Endocrine System Physiology; Stem Cells Research; Transcriptomics

Summary Retinal stem cells (RSCs) are rare pigmented cells found in the pigmented ciliary layer of the mammalian retina. Studies show that RSCs can replicate to maintain the stem cell pool and produce retinal progenitors that differentiate into all retinal cell types. We classified RSCs based on their level and distribution of pigment into heavily pigmented (HP), lightly pigmented (LP), and centrally pigmented (CP) spheres. We report that CP spheres are capable of generating large cobblestone lawns of retinal pigment epithelial (RPE) cells. The other clonal sphere types (HP and LP) primarily produce cells with neural morphology and fewer RPE cells. The RSCs are homogeneous, but their downstream progenitors are different. We found that CP spheres contain highly proliferative populations of early RPE progenitors that respond to proliferative signals from the surrounding non-pigmented cells. HP and LP spheres contain late RPE progenitors which are not affected by proliferative signals., Graphical abstract, Highlights • Three types of clonal retinal stem cell spheres form from the same single stem cell • Centrally pigmented spheres contain populations of early RPE progenitors • Heavily and lightly pigmented spheres contain populations of late RPE progenitors • Downstream RPE progenitors are different due to extrinsic and intrinsic factors, Biological sciences; Cell biology; Stem cells research

Summary The recruitment of Polycomb repressive complex 2 (PRC2) to gene promoters is critical for its function in repressing gene expression in murine embryonic stem cells (mESCs). However, previous studies have demonstrated that although the expression of early lineage-specific genes is largely repressed, the genome-wide PRC2 occupancy is unexpectedly reduced in naive mESCs. In this study, we provide evidence that fibroblast growth factor/extracellular signal-regulated kinase signaling determines the global PRC2 occupancy through regulating the expression of PRC2-recruiting factor JARID2 in naive mESCs. At the transcriptional level, the de-repression of bivalent genes is predominantly determined by the presence of cell signaling-associated transcription factors but not the status of PRC2 occupancy at gene promoters. Hence, this study not only reveals a key molecular mechanism by which cell signaling regulates the PRC2 occupancy in mESCs but also elucidates the functional roles of transcription factors and Polycomb-mediated epigenetic mechanisms in transcriptional regulation., Graphical Abstract, Highlights • FGF/ERK signaling positively regulates Jarid2 expression in mESCs • Reduced JARID2 causes global reduction of PRC2 occupancy in naive mESCs • Reduced PRC2 occupancy alone is insufficient to induce transcriptional activation • Cell signaling-associated transcription factors drive bivalent gene expression, Molecular Biology; Cell Biology; Stem Cells Research; Developmental Biology