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Embryonic stem cells can replicate continuously in the absence of senescence and,therefore,are immortal in culture. Although genome stability is essential for the survival of stem cells,proteome stability may have an equally important role in stem-cell identity and function. Furthermore,with the asymmetric divisions invoked by stem cells,the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. Therefore,a firm understanding of how stem cells maintain their proteome is of central importance. Here we show that human embryonic stem cells (hESCs) exhibit high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11 (known as RPN-6 in Caenorhabditis elegans) and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. FOXO4,an insulin/insulin-like growth factor-I (IGF-I) responsive transcription factor associated with long lifespan in invertebrates,regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Proteasome inhibition in hESCs affects the expression of pluripotency markers and the levels of specific markers of the distinct germ layers. Our results suggest a new regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates to hESC function and identity. View Publication

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Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes,for pharmacologic studies and as a potential resource for therapeutic transplant. To date,limited in vivo models exist for human intestine,all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here,we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme,both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme,as demonstrated by differentiated intestinal cell lineages (enterocytes,goblet cells,Paneth cells,tuft cells and enteroendocrine cells),presence of functional brush-border enzymes (lactase,sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore,transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection,suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology,disease and translational studies. View Publication

BACKGROUND: Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).backslashnbackslashnMETHODS AND RESULTS: To induce endothelial cell differentiation,undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days,CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation,these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%),CD31 (97.2+/-2.5%),and VE-cadherin (93.7+/-2.8%),form vascular-like channels,and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward,5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly,bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally,in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).backslashnbackslashnCONCLUSIONS: This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs,and enhance their angiogenic effects in vivo. Furthermore,activation of nAChRs has anti-apoptotic,angiogenic,and proliferative effects through MAPK and Akt signaling pathways. View Publication

Complement receptor 2-negative (CR2/CD21(-)) B cells have been found enriched in patients with autoimmune diseases and in common variable immunodeficiency (CVID) patients who are prone to autoimmunity. However,the physiology of CD21(-/lo) B cells remains poorly characterized. We found that some rheumatoid arthritis (RA) patients also display an increased frequency of CD21(-/lo) B cells in their blood. A majority of CD21(-/lo) B cells from RA and CVID patients expressed germline autoreactive antibodies,which recognized nuclear and cytoplasmic structures. In addition,these B cells were unable to induce calcium flux,become activated,or proliferate in response to B-cell receptor and/or CD40 triggering,suggesting that these autoreactive B cells may be anergic. Moreover,gene array analyses of CD21(-/lo) B cells revealed molecules specifically expressed in these B cells and that are likely to induce their unresponsive stage. Thus,CD21(-/lo) B cells contain mostly autoreactive unresponsive clones,which express a specific set of molecules that may represent new biomarkers to identify anergic B cells in humans. View Publication

One of the factors that can impact human embryonic stem cell expansion in stirred microcarrier culture reactors is mechanical stress caused by agitation. Therefore,we have investigated the effects of agitation on human embryonic stem cell growth and expression of pluripotent markers. Agitation of HES-2 cell line in microcarrier cultures in stirred spinner and agitated six-well plates did not affect expression of pluripotent markers,cell viability,and cell doubling times even after seven passages. However,HES-3 cell line was found to be shear sensitive,showing downregulation of three pluripotent markers Oct-4,mAb 84,and Tra-1-60,and lower cell densities in agitated as compared with static cultures,even after one passage. Cell viability was unaffected. The HES-3-agitated cultures showed increased expression of genes and proteins of the three germ layers. We were unable to prevent loss of pluripotent markers or restore doubling times in agitated HES-3 microcarrier cultures by addition of five different known cell protective polymers. In addition,the human induced pluripotent cell line IMR90 was also shown to differentiate in agitated conditions. These results indicate that the effect of agitation on cell growth and differentiation is cell line specific. We assume that the changes in the growth and differentiation of the agitation-sensitive (HES-3) cell line do not result from the effect of shear stress directly on cell viability,but rather by signaling effects that influence the cells to differentiate resulting in slower growth. View Publication

AIM Human embryonic stem cells (hESCs) represent a novel cell source to treat diseases such as heart failure and for use in drug screening. In this study,we aim to promote efficient generation of cardiomyocytes from hESCs by combining the current optimal techniques of controlled growth of undifferentiated cells and specific induction for cardiac differentiation. We also aim to examine whether these methods are scalable and whether the differentiated cells can be cryopreserved. METHODS & RESULTS hESCs were maintained without conditioned medium or feeders and were sequentially treated with activin A and bone morphogenetic protein-4 in a serum-free medium. This led to differentiation into cell populations containing high percentages of cardiomyocytes. The differentiated cells expressed appropriate cardiomyocyte markers and maintained contractility in culture,and the majority of the cells displayed working chamber (atrial and ventricular) type electrophysiological properties. In addition,the cell growth and differentiation process was adaptable to large culture formats. Moreover,the cardiomyocytes survived following cryopreservation,and viable cardiac grafts were detected after transplantation of cryopreserved cells into rat hearts following myocardial infarctions. CONCLUSION These results demonstrate that cardiomyocytes of high quality can be efficiently generated and cryopreserved using hESCs maintained in serum-free medium,a step forward towards the application of these cells to human clinical use or drug discovery. View Publication

AIMS: Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes. METHODS AND RESULTS: The hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice,readily formed embryoid bodies,and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors,structural,and ion channel genes. Furthermore,the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins,confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic dose-response (0.1-10 µM) to isoprenaline and Bay K 8644. Furthermore,carbamycholine (5 µM) suppressed the response to isoprenaline,while verapamil (2.5 µM) blocked Bay K 8644-induced inotropic activity. Moreover,verapamil (1 µM) reduced the corrected field potential duration by 45%,tetrodotoxin (10 µM) shortened the minimal field potential by 40%,and E-4031 (50 nM) prolonged field repolarization. CONCLUSION: Virus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular,structural,and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results,coupled with the potential to generate patient-specific hiPSC lines,hold great promise for the development of an in vitro platform for drug pharmacogenomics,disease modelling,and regenerative medicine. View Publication

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells,capable of germline transmission,from mouse somatic cells by transduction of four defined transcription factors. Here,we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4,and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology,proliferation,surface antigens,gene expression,epigenetic status of pluripotent cell-specific genes,and telomerase activity. Furthermore,these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts. View Publication

A limited number of accessible and representative models of human trophoblast cells currently exist for the study of placentation. Current stem cell models involve either a transition through a naïve stem cell state or precise dynamic control of multiple growth factors and small-molecule cues. Here,we demonstrated that a simple five-day treatment of human induced pluripotent stem cells with two small molecules,retinoic acid (RA) and Wnt agonist CHIR 99021 (CHIR),resulted in rapid,synergistic upregulation of CDX2. Transcriptomic analysis of RA + CHIR-treated cells showed high similarity to primary trophectoderm cells. Multipotency was verified via further differentiation towards cells with syncytiotrophoblast or extravillous trophoblast features. RA + CHIR-treated cells were also assessed for the established criteria defining a trophoblast cell model,and they possess all the features necessary to be considered valid. Collectively,our data demonstrate a facile,scalable method for generating functional trophoblast-like cells in vitro to better understand the placenta. View Publication

Background?-catenin,acting as the core effector of canonical Wnt signaling pathway,plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models,the ?-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood,partly due to the rapid and complex cell fate transitions during early differentiation.ResultsIn this study,we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of ?-catenin. Our analysis showed that a truncated ?-catenin lacking both N- and C-terminal domains (?N148C) could robustly rescue the DE formation,whereas hyperactive ?-catenin mutants with S33Y mutation or N-terminal deletion (?N90) had limited ability to induce DE lineage. Notably,the ?N148C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak ?-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes,whereas the hyperactive ?-catenin mutants activated mesoderm genes.ConclusionOur study uncovered an unconventional regulatory function of ?-catenin through weak transactivation,indicating that the levels of ?-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01279-5. View Publication

Gene edited human pluripotent stem cells are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive immune responses,but have largely not addressed the innate immune cells,such as neutrophils,that mediate inflammation and rejection processes occurring early after graft transplantation. We identify the adhesion molecule ICAM-1 as a hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In our experiments,we find that ICAM-1 blocking or knockout in human pluripotent stem cell-derived cardiovascular therapies imparts significantly diminished binding of multiple immune cell types. ICAM-1 knockout results in diminished T cell proliferation and activation responses in vitro and in longer in vivo retention/protection of knockout grafts following immune cell encounter in NeoThy humanized mice. We also introduce the ICAM-1 knockout edit into existing first-generation hypoimmune human pluripotent stem cells and prevent immune cell binding. This promising hypoimmune editing strategy has the potential to improve transplantation outcomes for regenerative therapies in the setting of cardiovascular pathologies and several other diseases. Hypoimmune gene editing in human pluripotent stem cells (hPSCs) provides a promising platform for cellular therapies. Here,the authors report that CRISPR mediated deletion of ICAM-1 in hPSC-derived grafts reduces immune cell adhesion,dampens T cell activation,and protects against immune rejection. View Publication