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Fragile X Syndrome (FXS), the leading monogenic cause of intellectual disability and autism spectrum disorder, is caused by expansion of a CGG trinucleotide repeat in the 5ʹ-UTR of the Fragile X Mental Retardation-1 (FMR1) gene. Epigenetic silencing of FMR1 results in loss of the Fragile X Mental Retardation Protein (FMRP). Although most studies to date have focused on excitatory neurons, recent evidence suggests that GABAergic inhibitory networks are also affected. To investigate human GABAergic neurogenesis, we established a method to reproducibly derive inhibitory neurons from multiple FXS and control human pluripotent stem cell (hPSC) lines. Electrophysiological analyses suggested that the developing FXS neurons had a delay in the GABA functional switch, a transition in fetal development that converts the GABAA channel’s function from depolarization to hyperpolarization, with profound effects on the developing brain. To investigate the cause of this delay, we analyzed 14 400 single-cell transcriptomes from FXS and control cells at 2 stages of GABAergic neurogenesis. While control and FXS cells were similar at the earlier time point, the later-stage FXS cells retained expression of neuroblast proliferation-associated genes and had lower levels of genes associated with action potential regulation, synapses, and mitochondria compared with controls. Our analysis suggests that loss of FMRP prolongs the proliferative stage of progenitors, which may result in more neurons remaining immature during the later stages of neurogenesis. This could have profound implications for homeostatic excitatory-inhibitory circuit development in FXS, and suggests a novel direction for understanding disease mechanisms that may help to guide therapeutic interventions.

Background Long-QT syndrome type 2 (LQT2) is a common malignant hereditary arrhythmia. Due to the lack of suitable animal and human models, the pathogenesis of LQT2 caused by human ether-a-go-go-related gene (hERG) deficiency is still unclear. In this study, we generated an hERG-deficient human cardiomyocyte (CM) model that simulates ‘human homozygous hERG mutations’ to explore the underlying impact of hERG dysfunction and the genotype–phenotype relationship of hERG deficiency. Methods The KCNH2 was knocked out in the human embryonic stem cell (hESC) H9 line using the CRISPR/Cas9 system. Using a chemically defined differentiation protocol, we obtained and verified hERG-deficient CMs. Subsequently, high-throughput microelectrode array (MEA) assays and drug interventions were performed to characterise the electrophysiological signatures of hERG-deficient cell lines. Results Our results showed that KCNH2 knockout did not affect the pluripotency or differentiation efficiency of H9 cells. Using high-throughput MEA assays, we found that the electric field potential duration and action potential duration of hERG-deficient CMs were significantly longer than those of normal CMs. The hERG-deficient lines also exhibited irregular rhythm and some early afterdepolarisations. Moreover, we used the hERG-deficient human CM model to evaluate the potency of agents (nifedipine and magnesium chloride) that may ameliorate the phenotype. Conclusions We established an hERG-deficient human CM model that exhibited QT prolongation, irregular rhythm and sensitivity to other ion channel blockers. This model serves as an important tool that can aid in understanding the fundamental impact of hERG dysfunction, elucidate the genotype–phenotype relationship of hERG deficiency and facilitate drug development.

Along with emergence of the organoids, their application in biomedical research has been currently one of the most fascinating themes. For the past few years, scientists have made significant contributions to deriving organoids representing the whole brain and specific brain regions. Coupled with somatic cell reprogramming and CRISPR/Cas9 editing, the organoid technologies were applied for disease modeling and drug screening. The methods to develop organoids further improved for rapid and efficient generation of cerebral organoids. Additionally, refining the methods to develop the regionally specified brain organoids enabled the investigation of development and interaction of the specific brain regions. Recent studies started resolving the issue in the lack of non-neuroectodermal cells in brain organoids, including vascular endothelial cells and microglia, which play fundamental roles in neurodevelopment and are involved in the pathophysiology of acute and chronic neural disorders. In this review, we highlight recent advances of neuronal organoid technologies, focusing on the region-specific brain organoids and complementation with endothelial cells and microglia, and discuss their potential applications to neuronal diseases.

BackgroundIn our previous work, we evaluated the therapeutic effects of 1α,25-Dihydroxyvitamin D3, the biologically active form of vitamin D, in the context of bleomycin-induced lung fibrosis. Contrary to the expected, vitamin D supplementation increased DNA damage expression and cellular senescence in alveolar epithelial type II cells and aggravated the overall lung pathology induced in mice by bleomycin. These effects were probably due to an alteration of the cellular DNA double-strand breaks repair capability. In the present work we have evaluated the effects of two hypocalcemic vitamin D analogs (calcipotriol and paricalcitol) in the expression of DNA damage in the context of minilungs derived from human embryonic stem cells and in the cell line A549.ResultsAs in the case of the cell line A549, bleomycin can induce DNA damage in the generated minilungs enriched in alveolar cells. The results indicate that, in contrast to vitamin D, the treatment of the minilungs with the hypocalcemic analogs reduce significantly the bulk of DNA damage expression in both bidimensional arrays of epithelial cells (2D minilungs) and lung bud organoids (3D minilungs). The initial evaluation of a battery of commercially available vitamin D analogs shows a significant reduction in A549 cells of gH2AFX expression levels, a marker of DNA damage, cell senescence and aging.ConclusionsThe treatments based in hypocalcemic vitamin D analogs might be used to reduce the bulk of DNA damage and eventually the subsequent cell senescence expression that underlie lung conditions as those that can evolve with fibrosis.

Background Thin endometrium (TE) is a challenging clinical issue in the reproductive medicine characterized by inadequate endometrial thickness, poor response to estrogen and no effective treatments currently. At present, the precise pathogenesis of thin endometria remains to be elucidated. We aimed to explore the related molecular mechanism of TE by comparing the transcriptome profiles of late-proliferative phase endometria between TE and matched controls. Methods We performed a bulk RNA-Seq (RNA-sequencing) of endometrial tissues in the late-proliferative phase in 7 TE and 7 matched controls for the first time. Differential gene expression analysis, gene ontology enrichment analysis and protein-protein interactions (PPIs) network analysis were performed. Immunohistochemistry was used for molecular expression and localization in endometria. Human endometrial stromal cells (HESCs) were isolated and cultured for verifying the functions of hub gene. Results Integrative data mining of our RNA-seq data in endometria revealed that most genes related to cell division and cell cycle were significantly inhibited, while inflammation activation, immune response and reactive oxygen species associated genes were upregulated in TE. PBK was identified as a hub of PPIs network, and its expression level was decreased by 2.43-fold in endometria of TE patients, particularly reduced in the stromal cells, which was paralleled by the decreased expression of Ki67. In vitro experiments showed that the depletion of PBK reduced the proliferation of HESCs by 50% and increased the apoptosis of HESCs by 1 time, meanwhile PBK expression was inhibited by oxidative stress (reduced by 76.2%), hypoxia (reduced by 51.9%) and inflammatory factors (reduced by approximately 50%). These results suggested that the insufficient expression of PBK was involved in the poor endometrial thickness in TE. Conclusions The endometrial transcriptome in late-proliferative phase showed suppressed cell proliferation in women with thin endometria and decreased expression of PBK in human endometrial stromal cells (HESCs), to which inflammation and reactive oxygen species contributed.

New technologies of induced pluripotent stem cells (iPSCs) and genome editing have emerged, allowing for the development of autologous transfusion therapies. We previously demonstrated definitive β‐globin production from human embryonic stem cell (hESC)‐derived erythroid cell generation via hemangioblast‐like ES‐sacs. In this study, we demonstrated normal β‐globin protein production from biallelic corrected sickle cell disease (SCD) iPSCs. We optimized our ES/iPS‐sac method for feeder cell‐free hESC maintenance followed by serum‐free ES‐sac generation, which is preferred for electroporation‐based genome editing. Surprisingly, the optimized protocol improved yields of ES‐sacs (25.9‐fold), hematopoietic‐like spherical cells (14.8‐fold), and erythroid cells (5.8‐fold), compared with our standard ES‐sac generation. We performed viral vector‐free gene correction in SCD iPSCs, resulting in one clone with monoallelic and one clone with biallelic correction, and using this serum‐free iPS‐sac culture, corrected iPSC‐generated erythroid cells with normal β‐globin, confirmed at DNA and protein levels. Our serum‐free ES/iPS‐sac protocol with gene correction will be useful to develop regenerative transfusion therapies for SCD., We have successfully corrected the sickle mutation in induced pluripotent stem cells (iPSCs) derived from a sickle cell disease patient using an improved serum‐free method for the generation of ES/iPS‐sacs, a hemangioblast‐like structure containing hematopoietic stem and progenitor cells that can be differentiated into erythroid cells containing mostly definitive globins, including γ‐ and β‐globins.

The new generation of organoids derived from human pluripotent stem cells holds a promising strategy for modeling host-bacteria interaction studies. Organoids recapitulate the composition, diversity of cell types, and, to some extent, the functional features of the native organ. We generated lung bud organoids derived from human embryonic stem cells to study the interaction of Streptococcus pneumoniae (pneumococcus) with the alveolar epithelium. Invasive pneumococcal disease is an important health problem that may occur as a result of the spread of pneumococcus from the lower respiratory tract to sterile sites. We show here an efficient experimental approach to model the main events of the pneumococcal infection that occur in the human lung, exploring bacterial adherence to the epithelium and internalization and triggering an innate response that includes the interaction with surfactant and the expression of representative cytokines and chemokines. Thus, this model, based on human minilungs, can be used to study pneumococcal virulence factors and the pathogenesis of different serotypes, and it will allow therapeutic interventions in a reliable human context. Importance: Streptococcus pneumoniae is responsible for high morbidity and mortalities rates worldwide, affecting mainly children and adults older than 65 years. Pneumococcus is also the most common etiologic agent of bacterial pneumonia and nonepidemic meningitis, and it is a frequent cause of bacterial sepsis. Although the introduction of pneumococcal vaccines has decreased the burden of pneumococcal disease, the rise of antibiotic-resistant strains and nonvaccine types by serotype replacement is worrisome. To study the biology of pneumococcus and to establish a reliable human model for pneumococcal pathogenesis, we generated human minilungs from embryonic stem cells. The results show that these organoids can be used to model some events occurring during the interaction of pneumococcus with the lung, such as adherence, internalization, and the initial alveolar innate response. This model also represents a great alternative for studying virulence factors involved in pneumonia, drug screening, and other therapeutic interventions. This work was supported by grant PI19CIII/00003 from the ISCIII to A.Z. and grantsPID2020-119298RB-I00 and PID2020-114546RB-I00 from the Spanish Ministry of Scienceand Innovation (MICINN) to J.Y. and O.Z., respectively. M.P.D.L. and J.M.R.-C. received grantsupport from AESI (PI20CIII/0029), the Spanish Association Against Cancer (AECC,CGB14143035THOM), and CIBERONC (group CB16/12/00273). S.A.R. was granted apostdoctoral fellowship by the Fundacão de Amparo á pesquisa do Estado do SãoPaulo (reference FAPESP-BEBE 2020/09919-0). Sí

Summary Pyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation., Graphical Abstract, Highlights • Exogenous pyruvate enhances TCA-cycle metabolism in hESCs • Exogenous pyruvate promotes BMP4-induced mesoderm differentiation • Exogenous pyruvate influences differentiation through AMPK/mTOR pathways, In this report, Chen and colleagues demonstrate that pyruvate promotes mesoderm differentiation in human embryonic stem cells. Pyruvate elevates TCA-cycle metabolism and modulates AMPK and mTOR pathways to promote BMP4-induced mesoderm differentiation. This discovery highlights that pyruvate as a common metabolite can regulate signaling pathways in cell-fate determination.

Summary GATA2 is essential for the endothelial-to-hematopoietic transition (EHT) and generation of hematopoietic stem cells (HSCs). It is poorly understood how GATA2 controls the development of human pluripotent stem cell (hPSC)-derived HS-like cells. Here, using human embryonic stem cells (hESCs) in which GATA2 overexpression was induced by doxycycline (Dox), we elucidated the dual functions of GATA2 in definitive hematopoiesis before and after the emergence of CD34+CD45+CD90+CD38– HS-like cells. Specifically, GATA2 promoted expansion of hemogenic precursors via the EHT and then helped to maintain HS-like cells in a quiescent state by regulating cell cycle. RNA sequencing showed that hPSC-derived HS-like cells were very similar to human fetal liver-derived HSCs. Our findings will help to elucidate the mechanism that controls the early stages of human definitive hematopoiesis and may help to develop a strategy to generate hPSC-derived HSCs., Graphical Abstract, Highlights • hESC-derived HS-like cells were highly similar to human fetal liver (FL) HSCs • Overexpression of GATA2 promoted expansion of hemogenic precursors through EHT • Overexpression of GATA2 enhanced the maintenance of HS-like cells • GATA2 overexpression maintained HS-like cells by inducing cell-cycle arrest, In this article, Professor Ma and his colleagues at IBT of CAMS & PUMC show that overexpression of GATA2 promoted expansion of hemogenic precursors through EHT, and thereafter enhanced the maintenance of HS-like cells in dormancy by cell-cycle arrest. hESC-derived HS-like cells were highly similar to human fetal liver (FL) HSCs.

Background Mesenchymal stem/stromal cells (MSCs) derived from human embryonic stem cells (hESCs) are attractive for their hematopoietic-supporting or potential therapeutic effects. However, procedures for high-effective and scalable generation of MSCs from hESCs within 2 weeks are still unestablished, which also hinder the development and mechanism study of mesengenesis. Methods In this study, we aimed to establish a strategy for programming hESC differentiation into MSCs by practicing small-scale chemical compound screening. Then, we used flow cytometry, multi-lineage differentiation, and karyotype analyses to investigate the biological phenotypes of the derived hESC-MSCs. Also, to explore whether the derived cells had hematopoietic-supporting ability in vitro, we carried out the cobblestone formation and megakaryocytic differentiation experiments. To further evaluate the function of hESC-MSCs in vivo, we transplanted the cells into a mouse model with hind limb ischemia. Results By simultaneous treatments with a JAK/STAT antagonist and a DNA methylation inhibitor, the efficiency of generating hESCs into CD73+ hESC-MPCs could reach 60% within 7 days. The derived cells further matured into hESC-MSCs, with comparable characteristics to those of adult MSCs in terms of surface markers, normal karyotype, and the potential for adipogenic, osteogenic, and chondrogenic differentiation. Functionally, hESC-MSCs had hematopoietic-supporting effects in vitro and could notably relieve symptoms of hind limb ischemia. Conclusions In the study, we established a high-efficient procedure for large-scale generation of MSCs from hESCs, which would be of great help for genesis and mechanism studies of MSCs. Meanwhile, the derived cells provide an alternative for translational clinical research. Electronic supplementary material The online version of this article (10.1186/s13287-019-1302-1) contains supplementary material, which is available to authorized users.

Spinal cord injury results in irreversible tissue damage and permanent sensorimotor impairment. The development of novel therapeutic strategies that improve the life quality of affected individuals is therefore of paramount importance. Cell transplantation is a promising approach for spinal cord injury treatment and the present study assesses the efficacy of human embryonic stem cell–derived neural crest cells as preclinical cell-based therapy candidates. The differentiated neural crest cells exhibited characteristic molecular signatures and produced a range of biologically active trophic factors that stimulated in vitro neurite outgrowth of rat primary dorsal root ganglia neurons. Transplantation of the neural crest cells into both acute and chronic rat cervical spinal cord injury models promoted remodeling of descending raphespinal projections and contributed to the partial recovery of forelimb motor function. The results achieved in this proof-of-concept study demonstrates that human embryonic stem cell–derived neural crest cells warrant further investigation as cell-based therapy candidates for the treatment of spinal cord injury.

Funder: Medical Research Council; doi: http://dx.doi.org/10.13039/501100000265, Funder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268, Funder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266, Background: A major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as good manufacturing practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited. Methods: We adapted research-grade protocols for the derivation and differentiation of long-term neuroepithelial stem cell progenitors (lt-NES) to GMP-grade reagents and factors suitable for clinical applications. We screened the robustness of the protocol with six clinical-grade hESC lines deposited in the UK Stem Cell Bank. Results: Here, we present a new GMP-compliant protocol to derive lt-NES, which are multipotent, bankable and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types. Conclusions: Overall, we report the feasibility of derivation and differentiation of clinical-grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.

MYB/c-MYB is a proto-oncogene encoding a helix-turn-helix transcription factor that plays a critical role in controlling proliferation and multilineage differentiation of hematopoietic progenitor and stem cells. Deregulation of MYB expression is associated with several types of leukemias and lymphomas. In an attempt to explore the role of the gene in the early human hematopoiesis, we have achieved bi-allelic targeting of MYB in human embryonic stem cells (hESCs) by TALEN-mediated homologous recombination. Furthermore, the gene targeting introduced eYFP Venus reporter gene into the MYB locus to delineate the expression pattern of MYB. The resulting two cell lines, WAe001-A-45 and WAe001-A-46, passed the standard assays for human pluripotent stem cells. Hematopoietic differentiation of these cell lines provides a model to study the role of MYB in human hematopoietic development.

Background The expression of a specific combination of transcription factors (TFs) in the multipotent progenitor cells (MPCs) is critical for determining pancreatic cell fate. NKX6.1 expression in PDX1+ MPCs is required for functional β cell generation. We have recently demonstrated the generation of a novel population of human pluripotent stem cell (hPSC)-derived MPCs that exclusively express NKX6.1, independently of PDX1 (PDX1−/NKX6.1+). Therefore, the aim of this study was to characterize this novel population to elucidate its role in pancreatic development. Methods The hPSCs were exposed to two differentiation protocols to generate MPCs that were analyzed using different techniques. Results Based on the expression of PDX1 and NKX6.1, we generated three different populations of MPCs, two of them were NKX6.1+. One of these NKX6.1 populations coexpressed PDX1 (PDX1+/NKX6.1+) which is known to mature into functional β cells, and an additional novel population did not express PDX1 (PDX1−/NKX6.1+) with an undefined role in pancreatic cell fate. This novel population was enriched using our recently established protocol, allowing their reorganization in three-dimensional (3D) structures. Since NKX6.1 induction in MPCs can direct them to endocrine and/or ductal cells in humans, we examined the coexpression of endocrine and ductal markers. We found that the expression of the pancreatic endocrine progenitor markers chromogranin A (CHGA) and neurogenin 3 (NGN3) was not detected in the NKX6.1+ 3D structures, while few structures were positive for NKX2.2, another endocrine progenitor marker, thereby shedding light on the origin of this novel population and its role in pancreatic endocrine development. Furthermore, SOX9 was highly expressed in the 3D structures, but cytokeratin 19, a main ductal marker, was not detected in these structures. Conclusions These data support the existence of two independent NKX6.1+ MPC populations during human pancreatic development and the novel PDX1−/NKX6.1+ population may be involved in a unique trajectory to generate β cells in humans. Electronic supplementary material The online version of this article (10.1186/s13287-018-0834-0) contains supplementary material, which is available to authorized users.

Background: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. Methods: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. Results: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. Conclusions: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory‐generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host‐to‐graft transfer of α‐synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α‐synuclein, in a clinical‐grade hESC line to generate SNCA +/− and SNCA −/− cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α‐synuclein preformed fibrils (PFFs) to seed the formation for Lewy‐like pathology as measured by phosphorylation of serine‐129 of α‐synuclein (pS129‐αSyn). Wild‐type neurons were fully susceptible to the formation of protein aggregates positive for pS129‐αSyn, while SNCA +/− and SNCA −/− neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n‐mediated gene editing confers a measure of resistance to Lewy pathology.

Given their totipotency, human embryonic stem cells (hESCs) can differentiate into all types of cells, including adipocytes, and provide an excellent research model for studying diseases associated with the metabolism of adipocytes, such as obesity and diabetes mellitus. Epigenetic regulation, including DNA methylation and histone modification, plays an essential role in the development and differentiation of hESCs. Lysine-specific demethylase 1 (LSD1), a well-characterized histone-modifying enzyme, demethylates dimethylated histone H3 lysine 4 (H3K4) through a flavin adenine dinucleotide (FAD)-dependent oxidative reaction. LSD1 affects the growth and differentiation of human and mouse ES cells, and the deletion of this gene in mice leads to embryonic lethality. Here, we investigated the functional role of LSD1 during the adipogenic differentiation of hESCs involving the demethylation of H3K4. We also found that treating hESCs with the LSD1 inhibitor CBB1007 promotes the adipogenic differentiation of hESCs.

Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects (CHDs). The right ventricular hypertrophy is associated with the survival rate of patients with repaired TOF. However, very little is known concerning its genetic etiology. Based on mouse model studies, a disintergrin and metalloprotease 10/17 (ADAM10 and ADAM17) are the key enzymes for the NOTCH and ErbB pathways, which are critical pathways for heart development. Mutations in these two genes have not been previously reported in human TOF patients. In this study, we sequenced ADAM10 and ADAM17 in a Han Chinese CHD cohort comprised of 80 TOF patients, 286 other CHD patients, and 480 matched healthy controls. Three missense variants of ADAM17 were only identified in 80 TOF patients, two of which (Y42D and L659P) are novel and not found in the Exome Aggregation Consortium (ExAC) database. Point mutation knock-in (KI) and ADAM17 knock-out (KO) human embryonic stem cells (hESCs) were generated by CRISPR/Cas9 and programmed to differentiate into cardiomyocytes (CMs). Y42D or L659P KI cells or complete KO cells all developed hypertrophy with disorganized sarcomeres. RNA-seq results showed that phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt), which is downstream of epidermal growth factor receptor (EGFR) signaling, was affected in both ADAM17 KO and KI hESC-CMs. In vitro experiments showed that these two mutations are loss-of-function mutations in shedding heparin-binding EGF-like growth factor (HB-EGF) but not NOTCH signaling. Our results revealed that CM hypertrophy in TOF could be the result of mutations in ADAM17 which affects HB-EGF/ErbB signaling.

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3β activity. Abbreviations: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III β-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium. Fil: Rodríguez Varela, Maria Soledad. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mucci, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina Fil: Videla Richardson, Guillermo Agustín. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina Fil: Morris Hanon, Olivia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina Fil: Furmento, Verónica Alejandra. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sevlever, Gustavo Emilio. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina Fil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina Fil: Romorini, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina

Expression of the constitutive androstane receptor (CAR, NR1I3) is enriched in the mature mammalian liver and increasingly recognized for its prominent role in regulating a myriad of processes including biotransformation, chemical transport, energy metabolism and lipid homeostasis. Previously, we demonstrated that CAR levels were markedly enhanced during the differentiation of hepatic-like cells derived from hESCs, prompting the hypothesis that CAR contributes a key functional role in directing human hepatogenesis. Here we demonstrate that over-expression of CAR in human embryonic stem cells (ESCs), transduced by a lentiviral vector, accelerates the maturation of hepatic-like cells, with CAR over-expressing cells exhibiting a 2.5-fold increase in albumin secretion by day 20 in culture differentiation, and significantly enhanced levels of mRNA expression of several liver-selective markers, including hepatic transcription factors, plasma proteins, biotransformation enzymes, and metabolic enzymes. CAR over-expressing cells also exhibited enhanced CITCO-inducible CYP3A7 enzymatic activity. Knockdown of CAR via siRNA attenuated the differentiation-dependent expression programs. In contrast, expression levels of the pregnane X receptor (PXR), a nuclear receptor most similar to CAR in primary sequence, were negligible in human fetal liver tissues or in the differentiating hESCs, and stable over-expression of PXR in hepatic-induced hESCs failed to enhance expression of hepatic phenotype markers. Together, these results define a novel role for human CAR in hepatic lineage commitment.

Background Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allograft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4+ T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. Results Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA−/− hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA−/− hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA−/− hESCs were powerless in IFN-γ inducible expression of HLA II. Conclusion We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA−/− hESCs can differentiate into tissue cells with non-HLA II expression. It’s promising that CIITA−/− hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors’ CD4+ T cells, which are the main effector cells of cellular immunity in allograft. Electronic supplementary material The online version of this article (doi:10.1186/s40659-015-0051-6) contains supplementary material, which is available to authorized users.

Spinal muscular atrophy (SMA), characterized by specific degeneration of spinal motor neurons, is caused by mutations in the survival of motor neuron 1, telomeric (SMN1) gene and subsequent decreased levels of functional SMN. How the deficiency of SMN, a ubiquitously expressed protein, leads to spinal motor neuron-specific degeneration in individuals affected by SMA remains unknown. In this study, we examined the role of SMN in mitochondrial axonal transport and morphology in human motor neurons by generating SMA type 1 patient-specific induced pluripotent stem cells (iPSCs) and differentiating these cells into spinal motor neurons. The initial specification of spinal motor neurons was not affected, but these SMA spinal motor neurons specifically degenerated following long-term culture. Moreover, at an early stage in SMA spinal motor neurons, but not in SMA forebrain neurons, the number of mitochondria, mitochondrial area and mitochondrial transport were significantly reduced in axons. Knocking down of SMN expression led to similar mitochondrial defects in spinal motor neurons derived from human embryonic stem cells, confirming that SMN deficiency results in impaired mitochondrial dynamics. Finally, the application of N-acetylcysteine (NAC) mitigated the impairment in mitochondrial transport and morphology and rescued motor neuron degeneration in SMA long-term cultures. Furthermore, NAC ameliorated the reduction in mitochondrial membrane potential in SMA spinal motor neurons, suggesting that NAC might rescue apoptosis and motor neuron degeneration by improving mitochondrial health. Overall, our data demonstrate that SMN deficiency results in abnormal mitochondrial transport and morphology and a subsequent reduction in mitochondrial health, which are implicated in the specific degeneration of spinal motor neurons in SMA., Summary: This study provides the first evidence in human models of spinal muscular atrophy of impaired mitochondrial dynamics, which serve as potential therapeutic targets for this devastating disease.

Stem cells can self-renew and differentiate into multiple cell types. These characteristics are maintained by the combination of specific signaling pathways and transcription factors that cooperate to establish a unique epigenetic state. Despite the broad interest of these mechanisms, the precise molecular controls by which extracellular signals organize epigenetic marks to confer multipotency remain to be uncovered. Here, we use human embryonic stem cells (hESCs) to show that the Activin–SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also self-renewal and differentiation. Finally, genetic studies in mice show that Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo, thereby confirming the existence of similar regulations in vivo during early embryonic development. Our results reveal the mechanisms by which extracellular factors coordinate chromatin status and cell fate decisions in hESCs.

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression., Summary: Human embryonic stem cell neural derivatives can be used to model the molecular and cellular properties of medulloblastoma.

Post-translational modifications (PTMs) are known to be essential mechanisms used by eukaryotic cells to diversify their protein functions and dynamically coordinate their signaling networks. Defects in PTMs have been linked to numerous developmental disorders and human diseases, highlighting the importance of PTMs in maintaining normal cellular states. Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are capable of self-renewal and differentiation into a variety of functional somatic cells; these cells hold a great promise for the advancement of biomedical research and clinical therapy. The mechanisms underlying cellular pluripotency in human cells have been extensively explored in the past decade. In addition to the vast amount of knowledge obtained from the genetic and transcriptional research in hPSCs, there is a rapidly growing interest in the stem cell biology field to examine pluripotency at the protein and PTM level. This review addresses recent progress toward understanding the role of PTMs (glycosylation, phosphorylation, acetylation and methylation) in the regulation of cellular pluripotency.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Summary The coordination of signalling pathways within the cell is vital for normal human development and post-natal tissue homeostasis. Gene expression and function is therefore tightly controlled at a number of levels. We investigated the role that post-translational modifications play during human hepatocyte differentiation. In particular, we examined the role of the small ubiquitin-like modifier (SUMO) proteins in this process. We used a human embryonic stem cell (hESC)-based model of hepatocyte differentiation to follow changes in protein SUMOylation. Moreover, to confirm the results derived from our cell-based system, we performed in vitro conjugation assays to characterise SUMO modification of a key liver-enriched transcription factor, HNF4α. Our analyses indicate that SUMOylation plays an important role during hepatocellular differentiation and this is mediated, in part, through regulation of the stability of HNF4α in a ubiquitin-dependent manner. Our study provides a better understanding of SUMOylation during human hepatocyte differentiation and maturation. Moreover, we believe the results will stimulate interest in the differentiation and phenotypic regulation of other somatic cell types.