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Purpose: To investigate the osteogenic potential of human embryonic stem cell-derived exosomes (hESC-Exos) and their effects on the differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs)., Methods: hESC-Exos were isolated and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. hUCMSCs were cultured with hESC-Exos to assess osteogenic differentiation through alizarin red staining, quantitative PCR (qPCR), and Western blotting. miRNA profiling of hESC-Exos was performed using miRNA microarray analysis. In vivo bone regeneration was evaluated using an ovariectomized rat model with bone defects treated with exosome-loaded scaffolds., Results: hESC-Exos significantly promoted the osteogenic differentiation of hUCMSCs, as evidenced by increased alizarin red staining and the upregulation of osteogenesis-related genes and proteins (ALP, RUNX2, OCN). miRNA analysis revealed that miR-21-5p is a key regulator that targets YAP1 and activates the Wnt/β-catenin signaling pathway. In vivo, hESC-Exos enhanced bone repair in ovariectomized rats, as demonstrated by increased bone mineral density and improved bone microarchitecture compared to those in controls., Conclusion: hESC-Exos exhibit significant osteogenic potential by promoting the differentiation of hUCMSCs and enhancing bone regeneration in vivo. This study revealed that the miR-21-5p-YAP1/β-catenin axis is a critical pathway, suggesting that the use of hESC-Exos is a promising therapeutic strategy for bone regeneration and repair., Competing Interests: The author(s) report no conflicts of interest in this work., (© 2024 Huang et al.)

The human CPAMD8 gene encodes proteins in the A2M/C3 (alpha-2-macroglobulin/complement 3) family, predominantly expressed in the distal tips of the retinal neuroepithelium that form the iris and ciliary body. Mutations in CPAMD8 have been linked to anterior segment dysplasia and congenital glaucoma. Using CRISPR/Cas9 editing, we inserted a 3*EAAAK-EGFP fluorescent tag into the CPAMD8 gene, enabling real-time observation of its expression and providing insights into its biological functions. The resulting gene-edited cell line retained normal stem cell morphology and karyotype, expressed essential pluripotency markers, and exhibited differentiation potential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Dopaminergic (DA) neurons play critical roles in various neurological processes and disorders, particularly Parkinson's disease. To enable precise visualization and tracking of DA neurons, we generated TH-EGFP, a tyrosine hydroxylase (TH)-driven enhanced green fluorescent protein (EGFP)-expressing knock-in cell line, by employing CRISPR/Cas9 technology. We introduced EGFP into the targeted genomic region of human embryonic stem cells (hESCs) and successfully established a TH-EGFP hESC line. Differentiation of TH-EGFP hESCs into human midbrain organoids confirmed the accurate integration of EGFP into TH-positive cells. The TH-EGFP hESC line serves as a valuable reporter for studying the development, maturation, and function of DA neurons., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

An ability to monitor cell cycle progression in real time has numerous applications in pluripotent stem cell-based models and therapeutics development. Fluorescence Ubiquitination-based Cell Cycle Indicator (FUCCI) systems enable live monitoring of the cell cycle in stem cells and their differentiated progenies in a non-invasive manner. We describe the generation and characterisation of a cell line with a doxycycline-inducible reporter system, iFUCCI, in the human embryonic stem cell (hESC) line MEL-1. MEL-1 iFUCCI hESCs exhibited a normal karyotype, expressed markers of the undifferentiated state, and demonstrated expected differentiation potential by giving rise to cell populations from all three germ layers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The MYH6 gene encodes α-myosin heavy chain in the adult human heart. MYH6 c.4034T > C (p.Leu1345Pro) mutation in MYH6 gene have been reported in patients with hypertrophic cardiomyopathy (HCM), but its causal role in HCM is less certain and has not been established unambiguously. Here, we generated a MYH6 (c.4034T > C) mutant human embryonic stem cell line (WAe009-A-1D) based on the CRISPR adenine base editing system that converts base A/T to G/C. The WAe009-A-1D cell maintains the morphology, pluripotency, and normal karyotype of the stem cells and is capable of differentiating into all three germ layers in vivo., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was financially supported by Science and Technology Research Project of Henan Provincial Department of Science and Technology (242102310355)., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The SFTPC gene is responsible for the production of the pulmonary surfactant protein C (SPC), a highly hydrophobic molecule that plays a crucial role in maintaining lung integrity through its influence on the synthesis of alveolar surfactant proteins. In this study, we harnessed the CRISPR/Cas9 system for precise genome editing to create a modified H1 human embryonic stem cell (hESC) line, incorporating the SFTPC-mCherry reporter construct. Therefore, the engineered SFTPC-mCherry knock-in (KI) hESC line can serve as an effective tool for tracking the expression patterns of the SFTPC gene as alveolar type 2 cells differentiate from hESCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Dilated Cardiomyopathy (DCM), a prevalent form of cardiomyopathy, is characterized by ventricular dilation and systolic dysfunction. Its etiology is intricate, encompassing multiple genetic and environmental elements. The LMOD2 (Leiomodin 2) gene has been demonstrated to be closely associated with the pathogenesis of DCM. In this study, a pure cell line was generated by knocking out the LMOD2 gene, and a DCM cell model was established through induced differentiation, thus providing a powerful experimental approach for further understanding the pathogenesis of DCM. It also provides a potential research orientation for the early diagnosis and individualized treatment of DCM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Calcium- and integrin-binding protein 1 (CIB1) has a diverse role in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. It is associated with cancer, cardiovascular disease and male infertility. Here, CRISPR/Cas9 genome-editing technology was employed to establish a CIB1 knockout human embryonic stem cell line, which exhibited normal pluripotency and karyotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Promoting endogenous cardiomyocyte proliferation is crucial for repairing infarcted hearts. Implantation of human pluripotent stem cell-derived cardiovascular progenitor cells (hCVPCs) promotes healing of infarcted hearts. However, little is known regarding their impact on host cardiomyocyte proliferation. Here, we revealed that hCVPC implantation into mouse infarcted hearts induced dedifferentiation and cell cycle re-entry of host cardiomyocytes, which was further confirmed in vitro by hCVPC-conditioned medium. Mechanistically, the PI3K/Akt signaling pathway mediated hCVPC-induced cardiomyocyte cell cycle re-entry. The findings reveal the novel function of hCVPCs in triggering cardiomyocyte dedifferentiation and cell cycle activation and highlight a strategy utilizing cells at early developmental stages to rejuvenate adult cardiomyocytes., Competing Interests: Declaration of competing interest There are no conflicts of interest to disclose., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Midbrain dopaminergic (mDA) neurons derived from human pluripotent stem cells (hPSCs) offer a promising cell source for cell replacement therapy in Parkinson's disease (PD). Single-cell RNA sequencing (scRNA-seq) of the developing human ventral midbrain has identified four cell types expressing markers used to define correctly patterned mDA progenitors. Here, we use CRISPR/Cas9 to generate a fluorescent human embryonic stem cell line for the isolation of two potential mDA progenitors, Rgl1 and ProgM. We expect that by isolating specific mDA progenitor cell type/s and defining their function, it may be possible to develop more precise cell replacement strategies for PD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Spinal and Bulbar Muscular Atrophy (SBMA) is an X-linked degenerative disorder of the neuromuscular system that is caused by an expanded CAG/polyglutamine (polyQ) tract within the Androgen Receptor (AR) gene. This mutation causes progressive muscle weakness and atrophy in men. Here, we report the establishment of the first SBMA disease-specific human embryonic stem cell (hESC) line in the NIH hESC registry, UM197-1. UM197-1 exhibits pluripotency, the ability to differentiate into three germ layers in vitro, and provides a new cellular model system to study SBMA disease pathogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

ID1 and ID3 are the members of the Inhibitor of DNA Binding (ID) protein family, which negatively regulates the basic helix-loop-helix (bHLH) transcription factors by forming heterodimers, are involved in neurodevelopment, cardiovascular development, and tumor metastasis. We created twoID1/3knockout cell lines from a human embryonic stem cell (hESC) line (H9) by CRISPR/Cas9-mediated gene targeting. These cell lines maintain stem cell morphology, a normal karyotype, and the expression of pluripotent marker genes. Additionally, they retain their in vivo differentiation potential. Thecell lines are valuable tools for studying the roles of ID1/3 in neurodevelopment and cardiovascular diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Activator protein 1 (AP-1) is involved in cell fate determination and function. To monitor the AP-1 activity, we cloned a AP-1 binding sites fragment into the upstream of minimal TATA-box promoter, then a luciferase-GFP reporter (LuciGFP) was designated to the downstream of the promoter. With CRISPR/Cas9, the AP-1-LuciGFP reporter was introduced into AAVS1 locus, a safe harbor for gene editing. Thus, this AP-1-LuciGFP reporter cell line could be subjected to monitor the AP-1 activity during the cell differentiation, cell fate transition and disease modeling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Over the past decade, the use of human stem cell-derived β cells (SC-β cells) to model pancreatic β cell development, function and disease has become increasingly common. Though protocols are rapidly improving, current directed differentiation strategies do not yield a pure population of insulin-positive SC-β cells in vitro. Therefore, it is experimentally advantageous to have reporter lines that allow for live sorting of insulin-positive populations. To aid in these studies, we have knocked mNeonGreen fluorescent protein into the endogenous insulin locus of the commonly used H1 (WA01) human embryonic stem cell line., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

CAR-NK cell therapy does not require HLA matching and has minimal side effects. However, traditional methods of engineering CARs into human tissue-derived NK cells exhibit heterogeneity, low transduction efficiency, and high manufacturing costs. Here, we provide a reliable approach for generating large-scale and cryopreserved mesothelin (MSLN) CAR-NK cells from human embryonic stem cells (hESCs) as an alternative cell source. We first constructed MSLN CAR-expressing hESCs to reduce CAR engineering costs and subsequently differentiated these stem cells into MSLN CAR-NK cells via an efficient organoid induction system. The MSLN CAR-NK cells exhibit the typical expression patterns of activating receptors, inhibitory receptors, and effector molecules of NK cells. In the presence of tumour cells, the MSLN CAR-NK cells show increased secretion of IFN-γ and TNF-α, as well as elevated CD107a expression level compared with induced NK cells. We cryopreserved the MSLN CAR-NK cells in liquid nitrogen using a clinical-grade freezing medium (CS10) for more than 6 months to mimic an off-the-shelf CAR-NK cell product. The thawed MSLN CAR-NK cells immediately recovered after 48-72-h culture and effectively eliminated ovarian tumour cells, including human primary ovarian tumour cells from patients. The thawed MSLN CAR-NK cells efficiently suppressed ovarian tumour development in vivo and prolonged the survival of tumour-bearing mice. Our study provides insights into the clinical translation of hESC-derived MSLN CAR-NK cells as a promising off-the-shelf cell product., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Background: Platelet transfusion therapy has made a great breakthrough in clinical practice, and the differentiation of human embryonic stem cells (hESCs) to produce functional platelets has become a new potential approach, however, efficient generation of functional platelets still faces great challenges. Here, we presented a novel approach to highly and efficiently generate mature megakaryocytes (MKs) and functional platelets from hESCs., Methods: In hypoxic conditions, we successfully replicated the maturation process of MKs and platelets in a controlled in vitro environment by introducing an optimal combination of cytokines at various stages of development. This method led to the generation of MKs and platelets derived from hESCs. Subsequently, mature MKs and functional platelets were further comprehensively investigated and characterized using a variety of methodologies, including flow cytometry analysis, RT-qPCR validation, Giemsa-Wright's staining, immunofluorescent staining, RNA transcriptome analysis, and DNA ploidy analysis. Additionally, the in vivo function of platelets was evaluated through the transplantation using thrombocytopenia model mice., Results: Under our 3D differentiation conditions with four sequential stages, hESCs could be efficiently induced into mature MKs, with 95% expressing CD41aCD42a or 90% expressing CD41aCD42b, and those MKs exhibited polyploid properties, produced filamentous proplatelet structures and further generated platelets. Furthermore, 95% of platelets showed CD42b + CD62p + phenotype upon the stimulation with ADP and TRAP-6, while 50% of platelets exhibited the ability to bind PAC-1, indicating that hESC-derived platelets possessed the in vitro functionality. In mice models of thrombocytopenia, hESC-derived platelets effectively restored hemostasis in a manner comparable to human blood-derived platelets. Further investigation on the mechanism of this sequential differentiation revealed that cellular differentiation and molecular interactions during the generation of hESC-derived MKs and platelets recapitulated the developmental trajectory of the megakaryopoiesis and thrombopoiesis., Conclusions: Thus, our results demonstrated that we successfully established a highly efficient differentiation of hESCs into mature MKs and functional platelets in vitro. The in vivo functionality of hESC-derived platelets closely resembles that of natural human platelets, thus offering a promising avenue for the development of functional platelets suitable for future clinical applications., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Ethics Committee of Guangzhou First people's Hospital (Approval No.: K-2021–008-01, and approval date: January 17, 2022.) to use umbilical cord blood and human platelets from healthy volunteers for Large-scale Preparation of Functional Platelets from hESCs/iPSCs. The patients or their guardians/legally authorized representatives provided written informed consent for participation in the study with the use of umbilical cord blood and human platelets. All mouse experiments were performed according to our experimental protocols approved by the Guangzhou Committee for the Use and Care of Laboratory Animals, and by the Animal Ethics Committee of South China University of Technology University (Approval number: 2019073, and approval date: December 12, 2019.). WiCell Research Institute has confirmed that there was the ethical approval for collection of human embryonic stem cells (WA01(H1) and WA09(H9)) with NIH Approval NIHhESC-10–0043 and NIH Approval NIHhESC-10–0062, and that the donors had signed informed consent ( www.wicell.org ). Competing  interests: The authors declare that there is no competing  interests., (© 2024. The Author(s).)

Gain of 1q is a highly recurrent chromosomal abnormality in human pluripotent stem cells. In this work, we show that gains of 1q impact the differentiation capacity to derivates of the three germ layers, leading to mis-specification to cranial placode and non-neural ectoderm during neuroectoderm differentiation. Also, we found a weaker expression of lineage-specific markers in hepatoblasts and cardiac progenitors. Competition assays show that the cells retain their selective advantage during differentiation, which is mediated by a higher expression of MDM4, a gene located in the common region of gain. MDM4 drives the winner phenotype of the mutant cells in both the undifferentiated and differentiating state by reducing the cells' sensitivity to DNA damage through decreased p53-mediated apoptosis. Finally, we found that cell density in culture plays a key role in promoting the competitive advantage of the cells by increasing DNA damage., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: This study was reviewed and approved by the Medical Ethics Committee UZ Brussel–VUB under protocol number B.U.N. 1432020000284 on 18th November 2020. The study was conducted in accordance with the ethical standards of the institutional research committee and ICH-GCP guidelines. Human embryonic stem cell lines were derived from embryos with the informed consent of the donating couple and the approval of the ethics committee at UZ Brussel., (© 2024. The Author(s).)

In mammals, early embryonic gastrulation process is high energy demanding. Previous studies showed that, unlike endoderm and mesoderm cells, neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process, which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/β-catenin signaling during neuroectoderm differentiation. Notably, histone lactylation, especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments, we further identified that four genes, including PAX6, were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition., Competing Interests: Declarations Ethical approval Not applicable. Consent to participate Not applicable. Consent to publish Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Alternative splicing (AS) alters the cis -regulatory landscape of mRNA isoforms, leading to transcripts with distinct localization, stability, and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESCs) and neural progenitor cells (NPCs) derived from the same ESCs. We performed de novo transcriptome assembly from high-confidence long reads from cytosolic, monosomal, light, and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Thousands of transcripts in each cell type exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 19% exhibited significant differential isoform sedimentation in ESCs and NPCs, respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated coding sequence (CDS) and untranslated region (UTR) lengths as important determinants of isoform-specific sedimentation profiles, and motif analyses reveal potential cell type-specific and subcellular fraction-associated RNA-binding protein signatures. Taken together, our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlight the utility of using a novel long-read sequencing-based method to study translational control., (© 2024 Ritter et al.; Published by Cold Spring Harbor Laboratory Press.)

Progress has been made in generating spinal cord and trunk derivatives from neuromesodermal progenitors (NMPs). However, maintaining the self-renewal of NMPs in vitro remains a challenge. In this study, we developed a cocktail of small molecules and growth factors that induces human embryonic stem cells to produce self-renewing NMPs (srNMPs) under chemically defined conditions. These srNMPs maintain the state of neuromesodermal progenitors in prolonged culture and have the potential to generate mesodermal cells and neurons, even at the single-cell level. Additionally, suspended srNMP aggregates can spontaneously differentiate into all tissue types of early embryonic trunks. Furthermore, transplanted srNMP-derived muscle satellite cells or progenitors of motor neurons were integrated into skeletal muscle or the spinal cord, respectively, and contributed to regeneration in mouse models. In summary, srNMPs hold great promise for applications in developmental biology and as renewable cell sources for cell therapy for trunk and spinal cord injuries., Competing Interests: Declaration of interests The authors have a patent application related to this work., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal disability and mortality. Although intensive studies and therapeutic approaches, there are limited restorative treatments till now. Human embryonic stem cell (hESCs)-derived cortical neural progenitors have shown great potentials in ischemic stroke in adult brain. However, it is unclear whether they are feasible for cortical reconstruction in immature brain with hypoxic-ischemic encephalopathy., Methods: By using embryonic body (EB) neural differentiation method combined with DAPT pre-treatment and quantitative cell transplantation, human cortical neuroblasts were obtained and transplanted into the cortex of hypoxic-ischemic injured brain with different dosages 2 weeks after surgery. Then, immunostaining, whole-cell patch clamp recordings and behavioral testing were applied to explore the graft survival and proliferation, fate commitment of cortical neuroblasts in vitro, neural circuit reconstruction and the therapeutic effects of cortical neuroblasts in HIE brain., Results: Transplantation of human cortical neural progenitor cells (hCNPs) in HIE-injured cortex exhibited long-term graft overgrowth. DAPT pre-treatment successfully synchronized hCNPs from different developmental stages (day 17, day 21, day 28) to deep layer cortical neuroblasts which survived well in HIE injured brain and greatly prevented graft overgrowth after transplantation. Importantly, the cortical neuroblasts primarily differentiated into deep-layer cortical neurons and extended long axons to their projection targets, such as the cortex, striatum, thalamus, and internal capsule in both ipsilateral and contralateral HIE-injured brain. The transplanted cortical neurons established synapses with host cortical neurons and exhibited spontaneous excitatory or inhibitory post-synaptic currents (sEPSCs or sIPSCs) five months post-transplantation. Rotarod and open field tests showed greatly improved animal behavior by intra-cortex transplantation of deep layer cortical neuroblasts in HIE injured brain., Conclusions: Transplanted hESCs derived cortical neuroblasts survive, project to endogenous targets, and integrate into host cortical neural circuits to rescue animal behavior in the HIE-injured brain without graft overgrowth, providing a novel and safe cell replacement strategy for the future treatment of HIE., Competing Interests: Declarations Ethics approval and consent to participate All animal studies were approved by the Experimental Ethics Committee at the School of Basic Medical Science, Fudan University (Approval No. 20220228-159). Tittle of the approved project: Stem cell therapy for brain disorder (Date of approval: Feb 28,2022). These experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The work has been reported in line with the ARRIVE guidelines 2.0 for the care and use of animals. Consent for publication Not applicable. Competing interests The authors declare no competing financial interests., (© 2024. The Author(s).)

Mapping the essential pathways for neuronal differentiation can uncover new therapeutics and models for neurodevelopmental disorders. We thus utilized a genome-wide loss-of-function library in haploid human embryonic stem cells, differentiated into caudal neuronal cells. We show that essential genes for caudal neurogenesis are enriched for secreted and membrane proteins and that a large group of neurological conditions, including neurodegenerative disorders, manifest early neuronal phenotypes. Furthermore, essential transcription factors are enriched with homeobox (HOX) genes demonstrating synergistic regulation and surprising non-redundant functions between HOXA6 and HOXB6 paralogs. Moreover, we establish the essentialome of imprinted genes during neurogenesis, demonstrating that maternally expressed genes are non-essential in pluripotent cells and their differentiated germ layers, yet several are essential for neuronal development. These include Beckwith-Wiedemann syndrome- and Angelman syndrome-related genes, for which we suggest a novel regulatory pathway. Overall, our work identifies essential pathways for caudal neuronal differentiation and stage-specific phenotypes of neurological disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Macular hole (MH) is a retinal break involving the fovea that causes impaired vision. Although advances in vitreoretinal surgical techniques achieve >90% MH closure rate, refractory cases still exist. For such cases, autologous retinal transplantation is an optional therapy showing good anatomic success, but visual improvement is limited and peripheral visual field defects are inevitable after graft harvesting. Here, using a non-human primate model, we evaluated whether human embryonic stem cell-derived retinal organoid (RO) sheet transplantation can be an effective option for treating MH. After transplantation, MH was successfully closed by continuous filling of the MH space with the RO sheet, resulting in improved visual function, although no host-graft synaptic connections were confirmed. Mild xeno-transplantation rejection was controlled by additional focal steroid injections and rod/cone photoreceptors developed in the graft. Overall, our findings suggest pluripotent stem cell-derived RO sheet transplantation as a practical option for refractory MH treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Organogenesis is a complex process that relies on a dynamic interplay between extrinsic factors originating from the microenvironment and tissue-specific intrinsic factors. For pancreatic endocrine cells, the local niche consists of acinar and ductal cells as well as neuronal, immune, endothelial, and stromal cells. Hematopoietic cells have been detected in human pancreas as early as 6 post-conception weeks, but whether they play a role during human endocrinogenesis remains unknown. To investigate this, we performed single-nucleus RNA sequencing (snRNA-seq) of the second-trimester human pancreas and identified a wide range of hematopoietic cells, including two distinct subsets of tissue-resident macrophages. Leveraging this discovery, we developed a co-culture system of human embryonic stem cell-derived endocrine-macrophage organoids to model their interaction in vitro. Here, we show that macrophages support the differentiation and viability of endocrine cells in vitro and enhance tissue engraftment, highlighting their potential role in tissue engineering strategies for diabetes., Competing Interests: Declaration of interests A.M., M.H.A., G.M.K., and M.C.N. are coinventors on one patent application related to this work. M.C.N. has a patent (WO2013163739A1) licensed to Sernova Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.

Human embryonic stem cells (hESCs) are naturally equipped to maintain genome integrity to minimize genetic mutations during early embryo development. However, genetic aberration risks and subsequent cellular changes in hESCs during in vitro culture pose a significant threat to stem cell therapy. While a few studies have reported specific somatic mutations and copy number variations (CNVs), the molecular mechanisms underlying the acquisition of 'culture-adapted phenotypes' by hESCs are largely unknown. Therefore, we conducted comprehensive genomic, single-cell transcriptomic, and single-cell ATAC-seq analyses of an isogenic hESC model displaying definitive 'culture-adapted phenotypes'. We found that hESCs lacking TP53, in which loss-of-function mutations were identified in human pluripotent stem cells (hPSCs), presented a surge in somatic mutations. Notably, hPSCs with a copy number gain of 20q11.21 during early passage did not present 'culture-adapted phenotypes' or BCL2L1 induction. Single-cell RNA-seq and ATAC-seq analyses revealed active transcriptional regulation at the 20q11.21 locus. Furthermore, the induction of BCL2L1 and TPX2 to trigger 'culture-adapted phenotypes' was associated with epigenetic changes facilitating TEA domain (TEAD) binding. These results suggest that 20q11.21 copy number gain and additional epigenetic changes are necessary for expressing 'culture-adapted phenotypes' by activating gene transcription at this specific locus., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Human prenatal skin is populated by innate immune cells, including macrophages, but whether they act solely in immunity or have additional functions in morphogenesis is unclear. Here we assembled a comprehensive multi-omics reference atlas of prenatal human skin (7-17 post-conception weeks), combining single-cell and spatial transcriptomics data, to characterize the microanatomical tissue niches of the skin. This atlas revealed that crosstalk between non-immune and immune cells underpins the formation of hair follicles, is implicated in scarless wound healing and is crucial for skin angiogenesis. We systematically compared a hair-bearing skin organoid (SkO) model derived from human embryonic stem cells and induced pluripotent stem cells to prenatal and adult skin 1 . The SkO model closely recapitulated in vivo skin epidermal and dermal cell types during hair follicle development and expression of genes implicated in the pathogenesis of genetic hair and skin disorders. However, the SkO model lacked immune cells and had markedly reduced endothelial cell heterogeneity and quantity. Our in vivo prenatal skin cell atlas indicated that macrophages and macrophage-derived growth factors have a role in driving endothelial development. Indeed, vascular network remodelling was enhanced following transfer of autologous macrophages derived from induced pluripotent stem cells into SkO cultures. Innate immune cells are therefore key players in skin morphogenesis beyond their conventional role in immunity, a function they achieve through crosstalk with non-immune cells., Competing Interests: Competing interests: J.L. and K.R.K., with the Indiana University Research and Technology Corporation, have a patent relating to the methodology and composition of SkOs (PCT/US2016/058174). K.R.K. is a consultant for StemCell Technologies. All other authors declare no competing interests., (© 2024. The Author(s).)

Objective: Human embryonic stem cell (hESC; SC)-derived pancreatic β cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCβ cells with varying degrees of maturation, these cells still differ from deceased donor human β cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCβ cell functional maturation., Methods: To monitor SCβ cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS 2A-EGFP/+ ;IAPP 2A-mScarlet/+ ) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations., Results: We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose., Conclusions: The INS 2A-EGFP/+ ;IAPP 2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived β cells in vitro. This tool has already been shared with 3 groups and is freely available to all., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cell replacement therapy is under development for dry age-related macular degeneration (AMD). A thin membrane resembling the Bruch's membrane is required to form a cell-on-membrane construct with retinal pigment epithelial (RPE) cells. These cells have been differentiated from human embryonic stem cells (hESCs) in vitro. A carrier membrane is required for cell implantation, which is biocompatible for cell growth and has dimensions and physical properties resembling the Bruch's membrane. Here a nanofiber electrospun poly-L-lactic acid (PLLA) membrane is tested for capacity to support cell growth and maturation. The requirements for laminin coating of the membrane are identified here. A porous electrospun nanofibrous PLLA membrane of ∼50 nm fiber diameter was developed as a prototype support for functional RPE cells grown as a monolayer. The need for laminin coating applied to the membrane following treatment with poly-L-ornithine (PLO), was identified in terms of cell growth and survival. Test membranes were compared in terms of hydrophilicity after laminin coating, mechanical properties of surface roughness and Young's modulus, porosity and ability to promote the attachment and proliferation of hESC-RPE cells in culture for up to 8 weeks. Over this time, RPE cell proliferation, morphology, and marker and gene expression, were monitored. The functional capacity of cell monolayers was identified in terms of transepithelial electrical resistance (TEER), phagocytosis of cells, as well as expression of the cytokines, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PLLA polymer fibers are naturally hydrophobic, so their hydrophilicity was improved by pretreatment with PLO for subsequent coating with the bioactive protein laminin. They were then assessed for amount of laminin adsorbed, contact angle and uniformity of coating using scanning electron microscopy (SEM). Pretreatment with 100% PLO gave the best result over 10% PLO treatment or no treatment prior to laminin adsorption with significantly greater surface stiffness and modulus. By 6 weeks after cell plating, the coated membranes could support a mature RPE monolayer showing a dense apical microvillus structure and pigmented 3D polygonal cell morphology. After 8 weeks, PLO (100%)-Lam coated membranes exhibited the highest cell number, cell proliferation, and RPE barrier function measured as TEER. RPE cells showed the higher levels of specific surface marker and gene expression. Microphthalmia-associated transcription factor expression was highly upregulated indicating maturation of cells. Functionality of cells was indicated by expression of VEGF and PEDF genes as well as phagocytic capacity. In conclusion, electrospun PLLA membranes coated with PLO-Lam have the physical and biological properties to support the distribution and migration of hESC-RPE cells throughout the whole structure. They represent a good membrane candidate for preparation of hESC-RPE cells as a monolayer for implantation into the subretinal space of AMD patients., (© 2024 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

More and more studies have demonstrated that pseudogenes possess coding ability, and the functions of their transcripts in the development of diseases have been partially revealed. However, the role of pseudogenes in maintenance of normal physiological states and life activities has long been neglected. Here, we identify pseudogenes that are dynamically expressed during human early embryogenesis, showing different expression patterns from that of adult tissues. We explore the expression correlation between pseudogenes and the parent genes, partly due to their shared gene regulatory elements or the potential regulation network between them. The essential role of three pseudogenes, PI4KAP1, TMED10P1, and FBXW4P1, in maintaining self-renewal of human embryonic stem cells is demonstrated. We further find that the three pseudogenes might perform their regulatory functions by binding to proteins or microRNAs. The pseudogene-related single-nucleotide polymorphisms are significantly associated with human congenital disease, further illustrating their importance during early embryonic development. Overall, this study is an excavation and exploration of functional pseudogenes during early human embryonic development, suggesting that pseudogenes are not only capable of being specifically activated in pathological states, but also play crucial roles in the maintenance of normal physiological states., Competing Interests: Conflict of interest The authors declare that no competing interests exist., (Copyright © 2024 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

N6-methyladenonsine (m6A) is ubiquitously distributed in mammalian mRNA. However, the precise involvement of m6A in early development has yet to be fully elucidated. Here, we report that deletion of the m6A demethylase ALKBH5 in human embryonic stem cells (hESCs) severely impairs definitive endoderm (DE) differentiation. ALKBH5-/- hESCs fail to undergo the primitive streak (PS) intermediate transition that precedes endoderm specification. Mechanistically, we show that ALKBH5 deficiency induces m6A hypermethylation around the 3' untranslated region (3'UTR) of GATA6 transcripts and destabilizes GATA6 mRNA in a YTHDF2-dependent manner. Moreover, GATA6 binds to the promoters of critical regulatory genes involved in Wnt/β-catenin signaling transduction, including the canonical Wnt antagonist DKK1 and DKK4, which are unexpectedly repressed upon the dysregulation of GATA6 mRNA metabolism. Remarkably, DKK1 and DKK4 both exhibit a pleiotropic effect in modulating the Wnt/β-catenin cascade and guard the endogenous signaling activation underlying DE formation as potential downstream targets of the ALKBH5-GATA6 regulation. Here, we unravel a role of ALKBH5 in human endoderm formation in vitro by modulating the canonical Wnt signaling logic through the previously unrecognized functions of DKK1/4, thus capturing a more comprehensive role of m6A in early human embryogenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Multiple Sclerosis (MS), inflammatory demyelinated lesions in the brain and spinal cord lead to neurodegeneration and progressive disability. Remyelination can restore fast saltatory conduction and neuroprotection but is inefficient in MS especially with increasing age, and is not yet treatable with therapies. Intrinsic and extrinsic inhibition of oligodendrocyte progenitor cell (OPC) function contributes to remyelination failure, and we hypothesised that the transplantation of 'improved' OPCs, genetically edited to overcome these obstacles, could improve remyelination. Here, we edit human(h) embryonic stem cell-derived OPCs to be unresponsive to a chemorepellent released from chronic MS lesions, and transplant them into rodent models of chronic lesions. Edited hOPCs display enhanced migration and remyelination compared to controls, regardless of the host age and length of time post-transplant. We show that genetic manipulation and transplantation of hOPCs overcomes the negative environment inhibiting remyelination, with translational implications for therapeutic strategies for people with progressive MS., (© 2024. The Author(s).)

Background: Lung injury and pulmonary fibrosis (PF), frequently arising as sequelae of severe and acute lung disease, currently face a dearth of effective therapeutic potions. Mesenchymal stem cells (MSCs) with immunomodulatory and tissue repair functions have immense potential to treat lung injury and PF. However, the optimal route of administration, timing, and frequency of dosing remain elusive. Human embryonic stem cell-derived immunity-and-matrix-regulatory cells (IMRCs) have shown therapeutic potential for lung injury and PF., Methods: To ascertain the optimal therapeutic regimen for IMRCs in PF, we conducted an experimental study. Utilizing a mouse model of PF induced by bleomycin (BLM), IMRCs were administered via either a single or double intravenous (IV) or intratracheal (IT) injection on the first and seventh days post-BLM induction., Results: Our findings revealed that IV infusion of IMRCs surpassed IT infusion in enhancing survival rates, facilitating body weight recovery, and optimizing Ashcroft and Szapiel scores among the model mice. Notably, IV administration exhibited a more profound ability to mitigate lung inflammation and fibrosis. Moreover, earlier and more frequent administrations of IMRCs were found to be advantageous in enhancing their therapeutic effects. Specifically, early administration with two IV infusions significantly improved body weight, lung organ coefficient, pulmonary ventilation and diffusion functions, and PF. This was accompanied by an increase in alveolar type I and II epithelial cells and a suppression of macrophage infiltration via CD24., Conclusion: Collectively, these results suggested that IMRCs infusion ameliorated lung injury by promoting lung regeneration and inhibiting macrophage infiltration in a route, time, and frequency-dependent manner., (© 2024. The Author(s).)

Mediator, a co-regulator complex required for RNA Polymerase II activity, interacts with tissue-specific transcription factors to regulate development and maintain homeostasis. We observe reduced Mediator subunit MED15 expression in endocrine hormone-producing pancreatic islets isolated from people living with type 2 diabetes and sought to understand how MED15 and Mediator control gene expression programs important for the function of insulin-producing β-cells. Here we show that Med15 is expressed during mouse β-cell development and maturation. Knockout of Med15 in mouse β-cells causes defects in β-cell maturation without affecting β-cell mass or insulin expression. ChIP-seq and co-immunoprecipitation analyses found that Med15 binds β-cell transcription factors Nkx6-1 and NeuroD1 to regulate key β-cell maturation genes. In support of a conserved role during human development, human embryonic stem cell-derived β-like cells, genetically engineered to express high levels of MED15, express increased levels of maturation markers. We provide evidence of a conserved role for Mediator in β-cell maturation and demonstrate an additional layer of control that tunes β-cell transcription factor function., (© 2024. The Author(s).)

The primed epiblast acts as a transitional stage between the relatively homogeneous naïve epiblast and the gastrulating embryo. Its formation entails coordinated changes in regulatory circuits driven by transcription factors and epigenetic modifications. Using a multi-omic approach in human embryonic stem cell models across the spectrum of peri-implantation development, we demonstrate that the transcription factors ZIC2 and ZIC3 have overlapping but essential roles in opening primed-specific enhancers. Together, they are essential to facilitate progression to and maintain primed pluripotency. ZIC2/3 accomplish this by recruiting SWI/SNF to chromatin and loss of ZIC2/3 or degradation of SWI/SNF both prevent enhancer activation. Loss of ZIC2/3 also results in transcriptome changes consistent with perturbed Polycomb activity and a shift towards the expression of genes linked to differentiation towards the mesendoderm. Additionally, we find an intriguing dependency on the transcriptional machinery for sustained recruitment of ZIC2/3 over a subset of primed-hESC specific enhancers. Taken together, ZIC2 and ZIC3 regulate highly dynamic lineage-specific enhancers and collectively act as key regulators of human primed pluripotency., (© 2024. The Author(s).)

Severe conjunctival damage can lead to extensive ocular cicatrisation, fornix shortening, and even ocular surface failure, resulting in significant vision impairment. Conjunctival reconstruction is the primary therapeutic strategy for these clinical conjunctival diseases. However, there have been limited studies on induced differentiation of conjunctival epithelial cells derived from stem cells. In this study, we established a chemical defined differentiation protocol from human embryonic stem cells (hESCs) into conjunctival epithelial cells. hES cell line H1 was used for differentiation, and RT-qPCR, immunofluorescence staining, Periodic-acid-Schiff staining (PAS), and transcriptome analysis were employed to identify the differentiated cells. Here, to imitate the development of the vertebrate conjunctiva, hESCs were induced using a three-step process involving first chetomin was used to induce ocular surface ectoderm, then nicotinamide was used to induce ocular surface epithelial progenitor cells, and finally epidermal growth factor, keratinocyte growth factor and other factors were used to differentiate mature conjunctival epithelial cells. hESC-derived conjunctival epithelial cells expressed mature conjunctival epithelial lineage markers (including PAX6, P63, K13). The presence of goblet cells was confirmed by positive PAS. Transcriptome analysis revealed that hESC-derived conjunctival epithelial cells possessed a more naïve phenotype, and exhibited greater proliferation capacity compared to mature human conjunctival epithelial cells, suggesting their potential as alternative seed cells for conjunctival reconstruction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, β-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME).  Data are available via ProteomeXchange with identifier PXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated., (© 2024 The Authors. PROTEOMICS published by Wiley‐VCH GmbH.)

Mutations in STAMBP have been well-established to cause congenital human microcephaly-capillary malformation (MIC-CAP) syndrome, a rare genetic disorder characterized by global developmental delay, severe microcephaly, capillary malformations, etc. Previous biochemical investigations and loss-of-function studies in mice have provided insights into the mechanism of STAMBP, however, it remains controversial how STAMBP deficiency leads to malformation of those affected tissues in patients. In this study, we investigated the function and underlying mechanism of STAMBP during neural differentiation of human embryonic stem cells (hESCs). We found that STAMBP is dispensable for the pluripotency maintenance or neural differentiation of hESCs. However, neural progenitor cells (NPCs) derived from STAMBP-deficient hESCs fail to be long-term maintained/expanded in vitro. We identified the anti-apoptotic protein CFLAR is down-regulated in those affected NPCs and ectopic expression of CFLAR rescues NPC defects induced by STAMBP-deficiency. Our study not only provides novel insight into the mechanism of neural defects in STAMBP mutant patients, it also indicates that the death receptor mediated apoptosis is an obstacle for long-term maintenance/expansion of NPCs in vitro thus counteracting this cell death pathway could be beneficial to the generation of NPCs in vitro., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Achaete-Scute Complex Homolog 1 (ASCL1) is a key regulator in the development and function of the nervous system, particularly in the process of neuronal and neuroendocrine cell differentiation. By employing the CRISPR/Cas9 system, we successfully established an ASCL1-mCherry knock-in human embryonic stem cell (hESC) line by inserting a P2A-mCherry fragment at the ASCL1 locus. The mCherry reporter effectively demonstrated the expression level of endogenous ASCL1 during the process of inducing pulmonary neuroendocrine cells (PNECs) from hESC. This reporter cell line holds significant value as a research tool for investigating the process of lung neuroendocrine cell differentiation, conducting drug screening, and exploring the underlying mechanisms of lung diseases associated with PNECs dysfunction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Deciphering the underlying gene regulatory networks (GRNs) that govern early human embryogenesis is critical for understanding developmental mechanisms yet remains challenging due to limited sample availability and the inherent complexity of the biological processes involved. To address this, we developed InPheRNo-ChIP, a computational framework that integrates multimodal data, including RNA-seq, transcription factor (TF)-specific ChIP-seq, and phenotypic labels, to reconstruct phenotype-relevant GRNs associated with endoderm development. The core of this method is a probabilistic graphical model that models the simultaneous effect of TFs on their putative target genes to influence a particular phenotypic outcome. Unlike the majority of existing GRN inference methods that are agnostic to the phenotypic outcomes, InPheRNo-ChIP directly incorporates phenotypic information during GRN inference, enabling the distinction between lineage-specific and general regulatory interactions. We integrated data from three experimental studies and applied InPheRNo-ChIP to infer the GRN governing the differentiation of human embryonic stem cells into definitive endoderm. Benchmarking against a scRNA-seq CRISPRi study demonstrated InPheRNo-ChIP's ability to identify regulatory interactions involving endoderm markers FOXA2, SMAD2, and SOX17, outperforming other methods. This highlights the importance of incorporating the phenotypic context during network inference. Furthermore, an ablation study confirms the synergistic contribution of ChIP-seq, RNA-seq, and phenotypic data, highlighting the value of multimodal integration for accurate phenotype-relevant GRN reconstruction., (© The Author(s) 2024. Published by Oxford University Press.)

It has been reported that human embryonic stem cells (hESCs) treated with BMP4 and inhibitors of TGFβ signaling (A83-01) and FGF signaling (PD173074), called BAP, can efficiently differentiate to extraembryonic (ExE) cells in vitro . Due to restricted access to human embryos, it is ethically impossible to test the developmental potential of ExE cells in vivo . Here, we demonstrate that most ExE cells expressed molecular markers for both trophoblasts (TBs) and amniotic cells (ACs). Following intra-uterine transplantation, ExE cells contributed to the mouse placenta. More interestingly, ExE cells could chimerize with the mouse blastocyst as, after injection into the blastocyst, they penetrated its trophectoderm. After implantation of the injected blastocysts into surrogate mice, human cells were found at E14 in placental labyrinth, junction zones, and even near the uterine decidua, expressed placental markers, and secreted human chorionic gonadotropin. Surprisingly, ExE cells also contributed to cartilages of the chimeric embryo with some expressing the chondrogenic marker SOX9, consistent with the mesodermal potential of TBs and ACs in the placenta. Deleting MSX2 , a mesodermal determinant, restricted the contribution of ExE cells to the placenta. Thus, we conclude that hESC-derived ExE cells can chimerize with the mouse blastocyst and contribute to both the placenta and cartilages of the chimera consistent with their heteogenious nature. Intra-uterus and intra-blastocyst injections are novel and sensitive methods to study the developmental potential of ExE cells., Competing Interests: Competing Interests: R.X. is a founder of ImStem Biotechnology, Inc., a stem cell company. The other authors declare no competing financial interests., (© The author(s).)

Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in β-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Thyroid carcinoma represents the first malignancy among the endocrine organs. Investigating the cellular hierarchy and the mechanisms underlying the initiation of thyroid carcinoma is crucial in thyroid cancer research. Here, we present a protocol for deriving thyroid cell lineage from human embryonic stem cells. We also describe steps for engineering thyroid progenitor cells utilizing CRISPR-Cas9 technology, which can be used to perform in vivo studies, thus facilitating the development of representative thyroid tumorigenesis models. For complete details on the use and execution of this protocol, please refer to Veschi et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Cardiovascular progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) are considered valuable cell sources for investigating cardiovascular physiology in vitro. Meeting the diverse needs of this application requires the large-scale production of CPCs in an in vitro environment. This study aimed to use an effective culture system utilizing signaling factors for the large-scale expansion of hESC-derived CPCs with the potential to differentiate into functional cardiac lineage cells., Methods and Results: Initially, CPCs were generated from hESCs using a 4-day differentiation protocol with a combination of four small molecules (CHIR99021, IWP2, SB-431542, and purmorphamine). These CPCs were then expanded and maintained in a medium containing three factors (bFGF, CHIR, and A83-01), resulting in a > 6,000-fold increase after 8 passages. These CPCs were successfully cryopreserved for an extended period in late passages. The expanded CPCs maintained their gene and protein expression signatures as well as their differentiation capacity through eight passages. Additionally, these CPCs could differentiate into four types of cardiac lineage cells: cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts, demonstrating appropriate functionality. Furthermore, the coculture of these CPC-derived cardiovascular lineage cells in rat tail collagen resulted in cardiac microtissue formation, highlighting the potential of this 3D platform for studying cardiovascular physiology in vitro., Conclusion: In conclusion, expandable hESC-derived CPCs demonstrated the ability to self-renewal and differentiation into functional cardiovascular lineage cells consistently across passages, which may apply as potential cell sources for in vitro cardiovascular studies., (© 2024. The Author(s).)

Background: Understanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule., Methods: To establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses., Results: We first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations., Conclusions: This study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis., (© 2024. The Author(s).)

During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential in regenerative medicine, offering new opportunities for personalized cell therapies. However, their clinical translation is hindered by the inevitable reliance on xenogeneic components in culture environments. This study addresses this challenge by engineering a fully synthetic, xeno-free culture substrate, whose surface composition is tailored systematically for xeno-free culture of hPSCs. A functional polymer surface, pGC2 (poly(glycidyl methacrylate-grafting-guanidine-co-carboxylic acrylate)), offers excellent cell-adhesive properties as well as non-cytotoxicity, enabling robust hESCs and hiPSCs growth while presenting cost-competitiveness and scalability over Matrigel. This investigation includes comprehensive evaluations of pGC2 across diverse experimental conditions, demonstrating its wide adaptability with various pluripotent stem cell lines, culture media, and substrates. Crucially, pGC2 supports long-term hESCs and hiPSCs expansion, up to ten passages without compromising their stemness and pluripotency. Notably, this study is the first to confirm an identical proteomic profile after ten passages of xeno-free cultivation of hiPSCs on a polymeric substrate compared to Matrigel. The innovative substrate bridges the gap between laboratory research and clinical translation, offering a new promising avenue for advancing stem cell-based therapies., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

The KCNQ1 gene encodes a voltage-gated potassium channel required for cardiac action potentials. Mutations in this gene have been associated with hereditary long QT syndrome 1, Jervell and Lange-Nielsen syndromes, and familial atrial fibrillation. The NM_000218.3(KCNQ1): c.604 + 2T > C mutation has been categorized as the causative variant leading to LQT1. In this study, we generated a KCNQ1 (c.644 + 2T > C) mutation human embryonic stem cell line WAe009-A-1L based on CRISPR base editing system. WAe009-A-1L cell has the potential to differentiate cardiomyocytes and would be used as an in vitro disease model for mechanism exploration and drug screening., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was financially supported by Henan Provincial Health Committee Foundation (LHGJ20210381)., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Human embryonic stem cells (hESCs) derived from blastocyst stage embryos present a primed state of pluripotency, whereas mouse ESCs (mESCs) display naïve pluripotency. Their unique characteristics make naïve hESCs more suitable for particular applications in biomedical research. This work aimed to derive hESCs from single blastomeres and determine their pluripotency state, which is currently unclear. We derived hESC lines from single blastomeres of 8-cell embryos and from whole blastocysts, and analysed several naïve pluripotency indicators, their transcriptomic profile and their trilineage differentiation potential. No significant differences were observed between blastomere-derived hESCs (bm-hESCs) and blastocyst-derived hESCs (bc-hESCs) for most naïve pluripotency indicators, including TFE3 localization, mitochondrial activity, and global DNA methylation and hydroxymethylation, nor for their trilineage differentiation potential. Nevertheless, bm-hESCs showed an increased single-cell clonogenicity and a higher expression of naïve pluripotency markers at early passages than bc-hESCs. Furthermore, RNA-seq revealed that bc-hESCs overexpressed a set of genes related to the post-implantational epiblast. Altogether, these results suggest that bm-hESCs, although displaying primed pluripotency, would be slightly closer to the naïve end of the pluripotency continuum than bc-hESCs., Competing Interests: Declaration of competing interest No competing interests declared., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)