Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells.

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.

FGF7 enhances the expression of ACE2 in human islet organoids aggravating SARS-CoV-2 infection.

The angiotensin-converting enzyme 2 (ACE2) is a primary cell surface viral binding receptor for SARS-CoV-2, so finding new regulatory molecules to modulate ACE2 expression levels is a promising strategy against COVID-19. In the current study, we utilized islet organoids derived from human embryonic stem cells (hESCs), animal models and COVID-19 patients to discover that fibroblast growth factor 7 (FGF7) enhances ACE2 expression within the islets, facilitating SARS-CoV-2 infection and resulting in impaired insulin secretion. Using hESC-derived islet organoids, we demonstrated that FGF7 interacts with FGF receptor 2 (FGFR2) and FGFR1 to upregulate ACE2 expression predominantly in ß cells. This upregulation increases both insulin secretion and susceptibility of ß cells to SARS-CoV-2 infection. Inhibiting FGFR counteracts the FGF7-induced ACE2 upregulation, subsequently reducing viral infection and replication in the islets. Furthermore, retrospective clinical data revealed that diabetic patients with severe COVID-19 symptoms exhibited elevated serum FGF7 levels compared to those with mild symptoms. Finally, animal experiments indicated that SARS-CoV-2 infection increased pancreatic FGF7 levels, resulting in a reduction of insulin concentrations in situ. Taken together, our research offers a potential regulatory strategy for ACE2 by controlling FGF7, thereby protecting islets from SARS-CoV-2 infection and preventing the progression of diabetes in the context of COVID-19.

SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss.

Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, because cells that have acquired specific genetic abnormalities experience a selective advantage in vitro. These abnormalities are highly recurrent in hPSC lines worldwide, but their functional consequences in differentiating cells are scarcely described. In this work, we show that the loss of chromosome 18q impairs neuroectoderm commitment and that downregulation of SALL3, a gene located in the common 18q loss region, is responsible for this failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, and transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, suggesting that these cells are in an altered state of pluripotency.

Generation of a DMD loss-of-function mutant human embryonic stem cell lines by CRISPR base editing.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder, which is caused mostly by frame-disrupting, out-of-frame variation in the dystrophin (DMD) gene. Loss-of- function mutations are the most common type of mutation in DMD, accounting for approximately 60-90% of all DMD variations. In this study, we used adenine base editing to generate a human embryonic stem cell line with splice-site mutations to mimic exon deletion variants in clinical Duchenne muscular dystrophy patients. This cell line has differentiation potential and a normal karyotypic.

Generation of a ISL1 homozygous knockout stem cell line (WAe009-A-1G) by episomal vector-based CRISPR/Cas9 system.

The ISL LIM homeobox 1 (ISL1) gene belongs to the LIM/homeodomain transcription factor family and plays a pivotal role in conveying multipotent and proliferative properties of cardiac precursor cells. Mutations in ISL1 are linked to congenital heart disease. To further explore ISL1's role in the human heart, we have created a homozygous ISL1 knockout (ISL1-KO) human embryonic stem cell line using the CRISPR/Cas9 system. Notably, this ISL1-KO cell line retains normal morphology, pluripotency, and karyotype. This resource serves as a valuable tool for investigating ISL1's function in cardiomyocyte differentiation.

Multiple treatments with human embryonic stem cell-derived mesenchymal progenitor cells preserved the fertility and ovarian function of perimenopausal mice undergoing natural aging.

OBJECTIVES: Currently, no approved stem cell-based therapies for preserving ovarian function during aging. To solve this problem, we developed a long-term treatment for human embryonic stem cell-derived mesenchymal progenitor cells (hESC-MPCs). We investigated whether the cells retained their ability to resist ovarian aging, which leads to delayed reproductive senescence. MATERIALS AND METHODS: In a middle-aged female model undergoing natural aging, we analyzed whether hESC-MPCs benefit the long-term maintenance of reproductive fecundity and ovarian reservoirs and how their transplantation regulates ovarian function. RESULTS: The number of primordial follicles and mice with regular estrous cycles were increased in perimenopausal mice who underwent multiple introductions of hESC-MPCs compared to age-matched controls. The estradiol levels in the hESC-MPCs group were restored to those in the young and adult groups. Embryonic development and live birth rates were higher in the hESC-MPC group than in the control group, suggesting that hESC-MPCs delayed ovarian senescence. In addition to their direct effects on the ovary, multiple-treatments with hESC-MPCs reduced ovarian fibrosis by downregulating inflammation and fibrosis-related genes via the suppression of myeloid-derived suppressor cells (MDSCs) produced in the bone marrow. CONCLUSIONS: Multiple introductions of hESC-MPCs could be a useful approach to prevent female reproductive senescence and that these cells are promising sources for cell therapy to postpone the ovarian aging and retain fecundity in perimenopausal women.

Advancements in Human Embryonic Stem Cell Research: Clinical Applications and Ethical Issues.

BACKGROUND: The development and use of human embryonic stem cells (hESCs) in regenerative medicine have been revolutionary, offering significant advancements in treating various diseases. These pluripotent cells, derived from early human embryos, are central to modern biomedical research. However, their application is mired in ethical and regulatory complexities related to the use of human embryos. METHOD: This review utilized key databases such as ClinicalTrials.gov, EU Clinical Trials Register, PubMed, and Google Scholar to gather recent clinical trials and studies involving hESCs. The focus was on their clinical application in regenerative medicine, emphasizing clinical trials and research directly involving hESCs. RESULTS: Preclinical studies and clinical trials in various areas like ophthalmology, neurology, endocrinology, and reproductive medicine have demonstrated the versatility of hESCs in regenerative medicine. These studies underscore the potential of hESCs in treating a wide array of conditions. However, the field faces ethical and regulatory challenges, with significant variations in policies and perspectives across different countries. CONCLUSION: The potential of hESCs in regenerative medicine is immense, offering new avenues for treating previously incurable diseases. However, navigating the ethical, legal, and regulatory landscapes is crucial for the continued advancement and responsible application of hESC research in the medical field. Considering both scientific potential and ethical implications, a balanced approach is essential for successfully integrating hESCs into clinical practice.

Isolation and Characterization of Extracellular Vesicles Through Orthogonal Approaches for the Development of Intraocular EV Therapy.

Purpose: Isolating extracellular vesicles (EVs) with high yield, replicable purity, and characterization remains a bottleneck in the development of EV therapeutics. To address these challenges, the current study aims to establish the necessary framework for preclinical and clinical studies in the development of stem cell-derived intraocular EV therapeutics. Methods: Small EVs (sEVs) were separated from the conditioned cell culture medium (CCM) of the human embryogenic stem cell-derived fully polarized retinal pigment epithelium (hESC-RPE-sEV) by a commercially available microfluidic tangential flow filtration (TFF) device ExoDisc (ED) or differential ultracentrifugation (dUC). The scaling and concentration capabilities and purity of recovered sEVs were assessed. Size, number, and surface markers of sEVs were determined by orthogonal approaches using multiple devices. Results: ED yielded higher numbers of sEVs, ranging from three to eight times higher depending on the measurement device, compared to dUC using the same 5 mL of CCM input. Within the same setting, the purity of ED-recovered hESC-RPE-sEVs was higher than that for dUC-recovered sEVs. ED yielded a higher concentration of particles, which is strongly correlated with the input volume, up to 10 mL (r = 0.98, P = 0.016). Meanwhile, comprehensive characterization profiles of EV surface markers between ED- and dUC-recovered hESC-RPE-sEVs were compatible. Conclusions: Our study supports TFF as a valuable strategy for separating sEVs for the development of intraocular EV therapeutics. However, there is a growing need for diverse devices to optimize TFF for use in EV preparation. Using orthogonal approaches in EV characterization remains ideal for reliably characterizing heterogeneous EV.

HAND factors regulate cardiac lineage commitment and differentiation from human pluripotent stem cells.

BACKGROUND: Transcription factors HAND1 and HAND2 (HAND1/2) play significant roles in cardiac organogenesis. Abnormal expression and deficiency of HAND1/2 result in severe cardiac defects. However, the function and mechanism of HAND1/2 in regulating human early cardiac lineage commitment and differentiation are still unclear. METHODS: With NKX2.5eGFP H9 human embryonic stem cells (hESCs), we established single and double knockout cell lines for HAND1 and HAND2, respectively, whose cardiomyocyte differentiation efficiency could be monitored by assessing NKX2.5-eGFP+ cells with flow cytometry. The expression of specific markers for heart fields and cardiomyocyte subtypes was examined by quantitative PCR, western blot and immunofluorescence staining. Microelectrode array and whole-cell patch clamp were performed to determine the electrophysiological characteristics of differentiated cardiomyocytes. The transcriptomic changes of HAND knockout cells were revealed by RNA sequencing. The HAND1/2 target genes were identified and validated experimentally by integrating with HAND1/2 chromatin immunoprecipitation sequencing data. RESULTS: Either HAND1 or HAND2 knockout did not affect the cardiomyocyte differentiation kinetics, whereas depletion of HAND1/2 resulted in delayed differentiation onset. HAND1 knockout biased cardiac mesoderm toward second heart field progenitors at the expense of first heart field progenitors, leading to increased expression of atrial and outflow tract cardiomyocyte markers, which was further confirmed by the appearance of atrial-like action potentials. By contrast, HAND2 knockout cardiomyocytes had reduced expression of atrial cardiomyocyte markers and displayed ventricular-like action potentials. HAND1/2-deficient hESCs were more inclined to second heart field lineage and its derived cardiomyocytes with atrial-like action potentials than HAND1 single knockout during differentiation. Further mechanistic investigations suggested TBX5 as one of the downstream targets of HAND1/2, whose overexpression partially restored the abnormal cardiomyocyte differentiation in HAND1/2-deficient hESCs. CONCLUSIONS: HAND1/2 have specific and redundant roles in cardiac lineage commitment and differentiation. These findings not only reveal the essential function of HAND1/2 in cardiac organogenesis, but also provide important information on the pathogenesis of HAND1/2 deficiency-related congenital heart diseases, which could potentially lead to new therapeutic strategies.

POZ/BTB and AT hook containing zinc finger 1 (PATZ1) suppresses differentiation and regulates metabolism in human embryonic stem cells.

Human embryonic stem cells (hESCs) can proliferate infinitely (self-renewal) and give rise to almost all types of somatic cells (pluripotency). Hence, understanding the molecular mechanism of pluripotency regulation is important for applications of hESCs in regenerative medicine. Here we report that PATZ1 is a key factor that regulates pluripotency and metabolism in hESCs. We found that depletion of PATZ1 is associated with rapid downregulation of master pluripotency genes and prominent deceleration of cell growth. We also revealed that PATZ1 regulates hESC pluripotency though binding the regulatory regions of OCT4 and NANOG. In addition, we demonstrated PATZ1 is a key node in the OCT4/NANOG transcriptional network. We further revealed that PATZ1 is essential for cell growth in hESCs. Importantly, we discovered that depletion of PATZ1 drives hESCs to exploit glycolysis which energetically compensates for the mitochondrial dysfunction. Overall, our study establishes the fundamental role of PATZ1 in regulating pluripotency in hESCs. Moreover, PATZ1 is essential for maintaining a steady metabolic homeostasis to refine the stemness of hESCs.

Scaling up GMP-grade dopaminergic cells for Parkinson's disease.

Stem cell therapy for Parkinson's disease requires demonstration of safety and efficacy of dopaminergic cells derived from a cell line, consideration of dose, and whether this is deliverable at scale. Park et al. demonstrate these requirements for a new hESC line and that their manufacturing methods allow for its scalability.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Decoding the universal human chromatin landscape through teratoma-based profiling.

Teratoma formation is key for evaluating differentiation of human pluripotent stem cells into embryonic germ layers and serves as a model for understanding stem cell differentiation and developmental processes. Its potential for insights into epigenome and transcriptome profiling is significant. This study integrates the analysis of the epigenome and transcriptome of hESC-generated teratomas, comparing transcriptomes between hESCs and teratomas. It employs cell type-specific expression patterns from single-cell data to deconvolve RNA-Seq data and identify cell types within teratomas. Our results provide a catalog of activating and repressive histone modifications, while also elucidating distinctive features of chromatin states. Construction of an epigenetic signature matrix enabled the quantification of diverse cell populations in teratomas and enhanced the ability to unravel the epigenetic landscape in heterogeneous tissue contexts. This study also includes a single cell multiome atlas of expression (scRNA-Seq) and chromatin accessibility (scATAC-Seq) of human teratomas, further revealing the complexity of these tissues. A histology-based digital staining tool further complemented the annotation of cell types in teratomas, enhancing our understanding of their cellular composition. This research is a valuable resource for examining teratoma epigenomic and transcriptomic landscapes and serves as a model for epigenetic data comparison.

Single-cell RNA sequencing reveals the role of mitochondrial dysfunction in the cardiogenic toxicity of perfluorooctane sulfonate in human embryonic stem cells.

Perfluorooctane sulfonate (PFOS), an endocrine-disrupting chemical pollutant, affects embryonic heart development; however, the mechanisms underlying its toxicity have not been fully elucidated. Here, Single-cell RNA sequencing (scRNA-seq) was used to investigate the overall effects of PFOS on myocardial differentiation from human embryonic stem cells (hESCs). Additionally, apoptosis, mitochondrial membrane potential, and ATP assays were performed. Downregulated cardiogenesis-related genes and inhibited cardiac differentiation were observed after PFOS exposure in vitro. The percentages of cardiomyocyte and cardiac progenitor cell clusters decreased significantly following exposure to PFOS, while the proportion of primitive endoderm cell was increased in PFOS group. Moreover, PFOS inhibited myocardial differentiation and blocked cellular development at the early- and middle-stage. A Gene Ontology analysis and pseudo-time trajectory illustrated that PFOS disturbed multiple processes related to cardiogenesis and oxidative phosphorylation in the mitochondria. Furthermore, PFOS decreased mitochondrial membrane potential and induced apoptosis. These results offer meaningful insights into the cardiogenic toxicity of PFOS exposure during heart formation as well as the adverse effects of PFOS on mitochondria.

Epigenetic repression of CHCHD2 enhances survival from single cell dissociation through attenuated Rho A kinase activity.

During in vitro culture, human pluripotent stem cells (hPSCs) often acquire survival advantages characterized by decreased susceptibility to mitochondrial cell death, known as "culture adaptation." This adaptation is associated with genetic and epigenetic abnormalities, including TP53 mutations, copy number variations, trisomy, and methylation changes. Understanding the molecular mechanisms underlying this acquired survival advantage is crucial for safe hPSC-based cell therapies. Through transcriptome and methylome analysis, we discovered that the epigenetic repression of CHCHD2, a mitochondrial protein, is a common occurrence during in vitro culture using enzymatic dissociation. We confirmed this finding through genetic perturbation and reconstitution experiments in normal human embryonic stem cells (hESCs). Loss of CHCHD2 expression conferred resistance to single cell dissociation-induced cell death, a common stress encountered during in vitro culture. Importantly, we found that the downregulation of CHCHD2 significantly attenuates the activity of Rho-associated protein kinase (ROCK), which is responsible for inducing single cell death in hESCs. This suggests that hESCs may survive routine enzyme-based cell dissociation by downregulating CHCHD2 and thereby attenuating ROCK activity. These findings provide insights into the mechanisms by which hPSCs acquire survival advantages and adapt to in vitro culture conditions.

Multifaceted Characterization of Human Embryonic Stem Cell-Derived Mesenchymal Stem/Stromal Cells Revealed Amelioration of Acute Liver Injury in NOD-SCID Mice.

Human embryonic stem cells (hESCs) are advantaged sources for large-scale and homogeneous mesenchymal stem/stromal cells (MSCs) generation. However, due to the limitations in high-efficiency procedures for hESC-MSCs induction, the systematic and detailed information of mesengenesis and early MSC development are largely obscure. In this study, we took advantage of the well-established twist-related protein 1 (TWIST1)-overexpressing hESCs and two small molecular cocktails (CHIR99021, decitabine) for high-efficient MSC induction. To assess the multidimensional biological and transcriptomic characteristics, we turned to cellular and molecular methods, such as flow cytometry (FCM), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in vitro tri-lineage differentiation, cytokine secretion analysis, in vivo transplantation for acute liver injury (ALI) management, and bioinformatics analyses (eg, gene ontology-biological processes [GO-BP], Kyoto Encyclopedia of Genes and Genomes [KEGG], HeatMap, and principal component analysis [PCA]). By combining TWIST1 overexpression (denoted as T) and the indicated small molecular cocktails (denoted as S), hESCs high-efficiently differentiated into MSCs (denoted as TS-MSCs, induced by T and S combination) within 2 weeks. TS-MSCs satisfied the criteria for MSC definition and revealed comparable tri-lineage differentiation potential and ameliorative efficacy upon ALI mice. According to RNA-sequencing (SEQ) analysis, we originally illuminated the gradual variations in gene expression pattern and the concomitant biofunctions of the programmed hESC-MSCs. Overall, our data indicated the feasibility of high-efficient generation of hESC-MSCs by TWIST1 and cocktail-based programming. The generated hESC-MSCs revealed multifaceted in vivo and in vitro biofunctions as adult BM-MSCs, which collectively suggested promising prospects in ALI management in future.

Enhanced quality of hESC-derived melanocytes through modified concentration of endothelin-1.

The study investigated the effectiveness of EDN1 and EDN3 cytokines in the differentiation of melanocytes from hESCs. The findings showed that 100 nM EDN1 was more effective in promoting hESC to CD117+/TYR+ melanoblasts compared to 100 nM EDN3. Additionally, maintaining melanoblasts is beneficial for preserving the ability to proliferate. The study found that 10 nM EDN1 helped maintain the proliferation of melanoblasts without over maturing them into melanocytes in the late stage of differentiation. Thus, using 100 nM EDN1 in the initial stage and 10 nM EDN1 in the late stage proved to be an efficient and cost-effective method for obtaining hESC-derived melanocytes. The preliminary results suggest that EDN1 promotes melanoblast formation during the initial differentiation stage through its binding to both the EDNRB receptor and EDNRA receptor. This study provides a valuable tool for studying the development of human melanocytes and modelling the biology of disease.

Divergent Roles of KLF4 During Primordial Germ Cell Fate Induction from Human Embryonic Stem Cells.

As a core transcriptional factor regulating pluripotency, Krüppel-like factor 4 (KLF4) has gained much attention in the field of stem cells during the past decades. However, few research have focused on the function of KLF4 during human primordial germ cell (PGC) specification. Here, we induced human PGC-like cells (hPGCLCs) from human embryonic stem cells (hESCs) and the derived hPGCLCs upregulated PGC-related genes, like SOX17, BLIMP1, TFAP2C, NANOS3, and the naïve pluripotency gene KLF4. The KLF4-knockout hESCs formed typical multicellular colonies with clear borders, expressed pluripotency genes, such as NANOG, OCT4, and SOX2, and exhibited no differences in proliferation capacity compared with wild type hESCs. Notably, KLF4 deletion in hESCs did not influence the induction of PGCLCs in vitro. In contrast, overexpression of KLF4 during PGC induction process inhibited the efficiency of PGCLC formation from hESCs in vitro. Overexpression of KLF4 may regenerate the naïve ground state in hESCs and results in repression for PGC specification. Thus, KLF4 could be a downstream target of human PGC program and the upregulation of KLF4 is prepared for late stage of germline development.

Effect of Valproic Acid on Promoting the Differentiation of Human Embryonic Stem Cells Into Cholangiocyte-Like Cells.

Cholangiocytes form a complex 3D network of bile ducts in the liver and contribute to liver function. The damage or destruction of cholangiocytes can lead to biliary diseases, and the shortage of cholangiocytes remains an obstacle for drug development targeting biliary diseases. Valproic acid (VPA) is a potent activator of Notch signaling pathway that is essential for cholangiocyte differentiation. Here, we report a VPA-based approach for cholangiocyte differentiation of human pluripotent stem cells. VPA activated Notch2 expression and upregulated HES-1, HEY-1, and Sox9 gene expression in hESC-derived hepatoblast. After 7 days treatment, VPA promoted successful differentiation of hepatoblast into cholangiocytes expressing cholangiocyte marker genes (AE2, AQP1, CFTR) and proteins (CK19 and CK7). In addition, the differentiated cholangiocytes formed bile duct-like structures after implantation into the spleen of NOD/SCID mice. Our results suggested that VPA can promote hESC differentiation to cholangiocyte-like cells. The induced cholangiocytes may serve as a potential cell source for both in vitro modeling and regenerative therapy of cholangiopathies. The findings can also support further development of small-molecule based differentiation protocols for cholangiocyte production.