Bright ferritin for long-term MR imaging of human embryonic stem cells.

BACKGROUND: A non-invasive imaging technology that can monitor cell viability, retention, distribution, and interaction with host tissue after transplantation is needed for optimizing and translating stem cell-based therapies. Current cell imaging approaches are limited in sensitivity or specificity, or both, for in vivo cell tracking. The objective of this study was to apply a novel ferritin-based magnetic resonance imaging (MRI) platform to longitudinal tracking of human embryonic stem cells (hESCs) in vivo. METHODS: Human embryonic stem cells (hESCs) were genetically modified to stably overexpress ferritin using the CRISPR-Cas9 system. Cellular toxicity associated with ferritin overexpression and manganese (Mn) supplementation were assessed based on cell viability, proliferation, and metabolic activity. Ferritin-overexpressing hESCs were characterized based on stem cell pluripotency and cardiac-lineage differentiation capability. Cells were supplemented with Mn and imaged in vitro as cell pellets on a preclinical 3 T MR scanner. T1-weighted images and T1 relaxation times were analyzed to assess contrast. For in vivo study, three million cells were injected into the leg muscle of non-obese diabetic severe combined immunodeficiency (NOD SCID) mice. Mn was administrated subcutaneously. T1-weighted sequences and T1 mapping were used to image the animals for longitudinal in vivo cell tracking. Cell survival, proliferation, and teratoma formation were non-invasively monitored by MRI. Histological analysis was used to validate MRI results. RESULTS: Ferritin-overexpressing hESCs labeled with 0.1 mM MnCl2 provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, proliferation, pluripotency, and differentiation into cardiomyocytes. Transplanted hESCs displayed significant bright contrast on MRI 24 h after Mn administration, with contrast persisting for 5 days. Bright contrast was recalled at 4-6 weeks with early teratoma outgrowth. CONCLUSIONS: The bright-ferritin platform provides the first demonstration of longitudinal cell tracking with signal recall, opening a window on the massive cell death that hESCs undergo in the weeks following transplantation before the surviving cell fraction proliferates to form teratomas.

Effects of Hypocalcemic Vitamin D Analogs in the Expression of DNA Damage Induced in Minilungs from hESCs: Implications for Lung Fibrosis.

In 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 the 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 in 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.

Generation of human embryonic stem cell-derived lung organoids.

This protocol describes how to generate lung organoids from human embryonic stem cells. Lung organoids form by self-assembly in Matrigel and contain lung epithelial cell types. The protocol presented in this study is simple and only uses 6 cytokines or small molecules. This protocol provides a promising tool to study human lung development, drug screening, regeneration, and disease modeling in vitro. For complete details on the use and execution of this protocol, please refer to Chen et al. (2018).

Matrix enrichment by black phosphorus improves ionization and reproducibility of mass spectrometry of intact cells, peptides, and amino acids.

Intact (whole) cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) is an established method for biotyping in clinical microbiology as well as for revealing phenotypic shifts in cultured eukaryotic cells. Intact cell MALDI-TOF MS has recently been introduced as a quality control tool for long-term cultures of pluripotent stem cells. Despite the potential this method holds for revealing minute changes in cells, there is still a need for improving the ionization efficiency or peak reproducibility. Here we report for the first time that supplementation by fine particles of black phosphorus to the standard MALDI matrices, such as sinapinic and α-cyano-4-hydroxycinnamic acids enhance intensities of mass spectra of particular amino acids and peptides, presumably by interactions with aromatic groups within the molecules. In addition, the particles of black phosphorus induce the formation of small and regularly dispersed crystals of sinapinic acid and α-cyano-4-hydroxycinnamic acid with the analyte on a steel MALDI target plate. Patterns of mass spectra recorded from intact cells using black phosphorus-enriched matrix were more reproducible and contained peaks of higher intensities when compared to matrix without black phosphorus supplementation. In summary, enrichment of common organic matrices by black phosphorus can improve discrimination data analysis by enhancing peak intensity and reproducibility of mass spectra acquired from intact cells.

Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant complex traits.

The hypothalamus regulates metabolic homeostasis by influencing behavior and endocrine systems. Given its role governing key traits, such as body weight and reproductive timing, understanding the genetic regulation of hypothalamic development and function could yield insights into disease pathogenesis. However, given its inaccessibility, studying human hypothalamic gene regulation has proven challenging. To address this gap, we generate a high-resolution chromatin architecture atlas of an established embryonic stem cell derived hypothalamic-like neuron model across three stages of in vitro differentiation. We profile accessible chromatin and identify physical contacts between gene promoters and putative cis-regulatory elements to characterize global regulatory landscape changes during hypothalamic differentiation. Next, we integrate these data with GWAS loci for various complex traits, identifying multiple candidate effector genes. Our results reveal common target genes for these traits, potentially affecting core developmental pathways. Our atlas will enable future efforts to determine hypothalamic mechanisms influencing disease susceptibility.

Global gene expression signatures in response to citrate-coated silver nanoparticles exposure.

Silver nanoparticles (AgNPs) are widely used in medical and commercial products for their unique antibacterial functions. However, the impact of AgNPs on human neural development is not well understood. To investigate the effect of AgNPs on human neural development, various doses of 20 nm citrate-coated AgNP (AgSC) were administered to human embryonic stem cell derived neural progenitors during the neuronal differentiation. Immunofluorescence staining with neuronal progenitor markers SOX2 (sex determining region Y-box 2) and Nestin (VI intermediate filament protein) showed that AgSC inhibited rosette formation, neuronal progenitor proliferation, and neurite outgrowth. Furthermore, AgSC promoted astrocyte activation and neuronal apoptosis. These adverse effects can be partially recovered with ascorbic acid. A genome-wide transcriptome analysis of both AgSC treated and untreated samples indicated that the most up-graduated genes were a group of Metallothionein (1F, 1E, 2A) proteins, a metal-binding protein that plays an essential role in metal homeostasis, heavy metal detoxification, and cellular anti-oxidative defence. The most significantly down-regulated genes were neuronal differentiation 6 (NEUROD6) and fork head box G1 (FOXG1). GO analyse indicated that the regulation of cholesterol biosynthetic process, neuron differentiation, synapse organization and pattern specification, oliogenesis, and neuronal apoptosis were the most impacted biological processes. KEGG pathway analyse showed that the most significantly impacted pathways were C5 isoprenoid, axon guidance, Notch, WNT, RAS-MAPK signalling pathways, lysosome, and apoptosis. Our data suggests that AgSCs interfered with metal homeostasis and cholesterol biosynthesis which induced oxidative stress, inhibited neurogenesis, axon guidance, and promoted apoptosis. Supplementation with ascorbic acid could act as an antioxidant to prevent AgSC-mediated neurotoxicity.

Human embryonic stem cell-derived cardiomyocyte platform screens inhibitors of SARS-CoV-2 infection.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated.

Zichong granules promote differentiation of ovarian granulosa-like cells from human embryonic stem cells in vitro.

OBJECTIVE: To investigate whether Zichong granules (, ZCKL), a very effective herbal formula for treating infertility, have an impact on the differentiation of ovarian granulosa cells from human embryonic stem cells (hESCs) in vitro, and to explore the cellular mechanisms of its clinical effects. METHODS: Serum from ZCKL-medicated rats was prepared and used to treat mesoderm cells derived from hESCs for 6 d. Normal rat serum and a set of growth factors were used as negative and positive controls, respectively. RESULTS: ZCKL-medicated rat serum, but not normal rat serum, induced hESCs-derived mesoderm cells to differentiate into functional ovarian granulosa-like cells (OGLCs) in a similar manner to defined growth factors. The induced OGLCs resembled the morphology of native human granulosa cells, expressed granulosa cell-specific markers at both the mRNA and protein levels, produced high levels of estradiol and strongly responded to follicle-stimulating hormone stimulation. Furthermore, mRNA levels of follistatin, mothers against decapentaplegic homolog 8 and bone morphogenetic protein 6 were dynamically changed during the process. CONCLUSION: In the ZCKL treatment of infertility, one mechanism by which ZCKL may act is by influencing ovarian granulosa cell differentiation and development, possibly through the follistatin and BMP/SMAD signaling pathways.

Network pharmacology and RNA-sequencing reveal the molecular mechanism of Xuebijing injection on COVID-19-induced cardiac dysfunction.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Up to 20%-30% of patients hospitalized with COVID-19 have evidence of cardiac dysfunction. Xuebijing injection is a compound injection containing five traditional Chinese medicine ingredients, which can protect cells from SARS-CoV-2-induced cell death and improve cardiac function. However, the specific protective mechanism of Xuebijing injection on COVID-19-induced cardiac dysfunction remains unclear. METHODS: The therapeutic effect of Xuebijing injection on COVID-19 was validated by the TCM Anti COVID-19 (TCMATCOV) platform. RNA-sequencing (RNA-seq) data from GSE150392 was used to find differentially expressed genes (DEGs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. Data from GSE151879 was used to verify the expression of Angiotensin I Converting Enzyme 2 (ACE2) and central hub genes in both human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) and adult human CMs with SARS-CoV-2 infection. RESULTS: A total of 97 proteins were identified as the therapeutic targets of Xuebijing injection for COVID-19. There were 22 DEGs in SARS-CoV-2 infected hiPSC-CMs overlapped with the 97 therapeutic targets, which might be the therapeutic targets of Xuebijing injection on COVID-19-induced cardiac dysfunction. Based on the bioinformatics analysis, 7 genes (CCL2, CXCL8, FOS, IFNB1, IL-1A, IL-1B, SERPINE1) were identified as central hub genes and enriched in pathways including cytokines, inflammation, cell senescence and oxidative stress. ACE2, the receptor of SARS-CoV-2, and the 7 central hub genes were differentially expressed in at least two kinds of SARS-CoV-2 infected CMs. Besides, FOS and quercetin exhibited the tightest binding by molecular docking analysis. CONCLUSION: Our study indicated the underlying protective effect of Xuebijing injection on COVID-19, especially on COVID19-induced cardiac dysfunction, which provided the theoretical basis for exploring the potential protective mechanism of Xuebijing injection on COVID19-induced cardiac dysfunction.

Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells.

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.

Carbon nanodot decorated acellular dermal matrix hydrogel augments chronic wound closure.

Impaired skin regeneration in chronic wounds like in diabetes corresponds to high oxidative stress, poor angiogenesis and insufficient collagen hyperplasia. Therefore, a multifaceted strategy for treatment is required to address critical issues associated with chronic wound healing. Fascinating application of nanomaterials in chronic wounds is still limited; hence, in the present work bioactive solubilized decellularized dermal matrix (sADM) was employed to form a hydrogel with chitosan (CTS) at physiological pH/temperature and modified with reactive oxygen species (ROS) scavenging carbon nanodots (ND). A detailed in vitro investigation found that the ND modified bioactive hydrogel (CsADMND) is suitable for human amniotic membrane derived stem cell (hAMSC) delivery. Also, CsADMND was observed to possess a good ROS scavenging property, hemocompatibility and pro-angiogenic potential as demonstrated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), haemolysis and chick chorioallantoic membrane (CAM) assay, respectively. The hybrid hydrogel promoted migration of cells in vitro in scratch assay owing to its antioxidant potential and the presence of bioactive moieties. Further, its efficacy in healing full thickness (FT) chronic wounds was evaluated in a streptozotocin (STZ) induced diabetic model. The CsADMND hydrogel after association with hAMSCs led to stimulation of early angiogenesis, superior collagen deposition, rapid wound closure, complete reepithelialisation, and formation of distinct organized dermal epidermal junctions (DEJ) post 21 days of healing. These results suggest that the hAMSC laden CsADMND hydrogel may serve as a promising therapeutic strategy for the management of chronic wounds.

Establishment of drug screening in human embryonic stem cells based on a high-content screening system.

High-content screening (HCS) systems can be used for high-throughput screening of drugs in human embryonic stem cells (hESCs). However, hESCs require immunofluorescence staining with stemness markers (e.g., Oct-4) prior to HCS, which can be time consuming and labor intensive. In this study, we employed transgenic hESCs with enhanced green fluorescent protein driven by stemness gene Oct-4 promoter (Oct-4-EGFP-H9), in which the colony area and relative green fluorescence area inferred a state of hESC proliferation and stemness, respectively. The Oct-4-EGFP-H9 transgenic hESCs were cultured in mTeSR medium with different concentrations of 5-Fluorouracil (5-FU), vitamin C (VC), or retinoic acid (RA) for 5-7 days, followed by repeated imaging using the HCS system. Finally, the hESC colony area and green fluorescence area were calculated. Results showed that 5-FU treatment markedly reduced colony area in a dose-dependent manner, whereas VC and RA treatments did not. MTT assay and flow cytometry indicated that 5-FU inhibited the proliferation of hESCs significantly, verifying reliability of the data from the HCS system based on colony area analysis. The green fluorescence to total colony area ratio decreased with RA treatment, suggesting that RA significantly promoted differentiation, whereas 5-FU and VC had almost no effect, as verified by quantitative real-time polymerase chain reaction and western blot analysis. In conclusion, our study established a rapid and efficient drug screening system without the requirement of staining based on HCS.

Targeted Delivery of Iron Oxide Nanoparticle-Loaded Human Embryonic Stem Cell-Derived Spherical Neural Masses for Treating Intracerebral Hemorrhage.

This study evaluated the potential of iron oxide nanoparticle-loaded human embryonic stem cell (ESC)-derived spherical neural masses (SNMs) to improve the transportation of stem cells to the brain, ameliorate brain damage from intracerebral hemorrhage (ICH), and recover the functional status after ICH under an external magnetic field of a magnet attached to a helmet. At 24 h after induction of ICH, rats were randomly separated into three experimental groups: ICH with injection of phosphate-buffered saline (PBS group), ICH with intravenous injection of magnetosome-like ferrimagnetic iron oxide nanocubes (FION)-labeled SNMs (SNMs* group), and ICH with intravenous injection of FION-labeled SNMs followed by three days of external magnetic field exposure for targeted delivery by a magnet-embedded helmet (SNMs*+Helmet group). On day 3 after ICH induction, an increased Prussian blue-stained area and decreased swelling volume were observed in the SNMs*+Helmet group compared with that of the other groups. A significantly decreased recruitment of macrophages and neutrophils and a downregulation of pro-inflammatory cytokines followed by improved neurological function three days after ICH were observed in the SNMs*+Helmet group. Hemispheric atrophy at six weeks after ICH was significantly decreased in the SNMs*+Helmet group compared with that of the PBS group. In conclusion, we have developed a targeted delivery system using FION tagged to stem cells and a magnet-embedded helmet. The targeted delivery of SNMs might have the potential for developing novel therapeutic strategies for ICH.

Human Embryonic Stem Cell-Derived Wilson's Disease Model for Screening Drug Efficacy.

Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have been extensively studied as an alternative cellular model for recapitulating phenotypic and pathophysiologic characters of human diseases. Particularly, hiPSCs generated from the genetic disease somatic cells could provide a good cellular model to screen potential drugs for treating human genetic disorders. However, the patient-derived cellular model has a limitation when the patient samples bearing genetic mutations are difficult to obtain due to their rarity. Thus, in this study, we explored the potential use of hPSC-derived Wilson's disease model generated without a patient sample to provide an alternative approach for modeling human genetic disease by applying gene editing technology. Wilson's disease hPSCs were generated by introducing a R778L mutation in the ATP7B gene (c.2333G>T) using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system into wildtype hESCs. Established Wilson's disease hESCs were further differentiated into hepatocyte-like cells (HLCs) and analyzed for disease phenotypes and responses against therapeutic agent treatment. R778L mutation in the ATP7B gene was successfully introduced into wildtype hESCs, and the introduction of the mutation neither altered the self-renewal ability of hESCs nor the differentiation capability into HLCs. However, R778L mutation-introduced HLCs exhibited higher vulnerability against excessive copper supplementation than wildtype HLCs. Finally, the applicability of the R778L mutation introduced HLCs in drug screening was further demonstrated using therapeutic agents against the Wilson's diseases. Therefore, the established model in this study could effectively mimic the Wilson's disease without patient's somatic cells and could provide a reliable alternative model for studying and drug screening of Wilson's disease.

PGC-1α activator ZLN005 promotes maturation of cardiomyocytes derived from human embryonic stem cells.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in biomedical applications. However, the immature state of cardiomyocytes obtained using existing protocols limits the application of hPSC-CMs. Unlike adult cardiac myocytes, hPSC-CMs generate ATP through an immature metabolic pathway-aerobic glycolysis, instead of mitochondrial oxidative phosphorylation (OXPHOS). Hence, metabolic switching is critical for functional maturation in hPSC-CMs. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is a key regulator of mitochondrial biogenesis and metabolism, which may help promote cardiac maturation during development. In this study, we investigated the effects of PGC-1α and its activator ZLN005 on the maturation of human embryonic stem cell-derived cardiomyocyte (hESC-CM). hESC-CMs were generated using a chemically defined differentiation protocol and supplemented with either ZLN005 or DMSO (control) on differentiating days 10 to 12. Biological assays were then performed around day 30. ZLN005 treatment upregulated the expressions of PGC-1α and mitochondrial function-related genes in hESC-CMs and induced more mature energy metabolism compared with the control group. In addition, ZLN005 treatment increased cell sarcomere length, improved cell calcium handling, and enhanced intercellular connectivity. These findings support an effective approach to promote hESC-CM maturation, which is critical for the application of hESC-CM in disease modeling, drug screening, and engineering cardiac tissue.

The importance of transporters and cell polarization for the evaluation of human stem cell-derived hepatic cells.

One of the most promising applications of human pluripotent stem cells is their utilization for human-based pharmacological models. Despite the fact that membrane transporters expressed in the liver play pivotal role in various hepatic functions, thus far only little attention was devoted to the membrane transporter composition of the stem cell-derived liver models. In the present work, we have differentiated HUES9, a human embryonic stem cell line, toward the hepatic lineage, and monitored the expression levels of numerous differentiation marker and liver transporter genes with special focus on ABC transporters. In addition, the effect of bile acid treatment and polarizing culturing conditions on hepatic maturation has been assessed. We found that most transporter genes crucial for hepatic functions are markedly induced during hepatic differentiation; however, as regards the transporter composition the end-stage cells still exhibited dual, hepatocyte and cholangiocyte character. Although the bile acid treatment and sandwich culturing only slightly influenced the gene expressions, the stimulated cell polarization resulted in formation of bile canaliculi and proper localization of transporters. Our results point to the importance of membrane transporters in human stem cell-derived hepatic models and demonstrate the relevance of cell polarization in generation of applicable cellular models with correctly localized transporters. On the basis of our observations we suggest that conventional criteria for the evaluation of the quality of stem cell-derived hepatocyte-like cells ought to be augmented with additional elements, such as polarized and functional expression of hepatic transporters.

A Preclinical Safety Study of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells for Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is a common disease trending towards epidemic proportions and is a leading cause of irreversible vision loss in people over the age of 65. A pathomechanism of AMD is death and/or dysfunction of retinal pigment epithelial (RPE) cells; RPE loss invariably results in photoreceptor atrophy. Treatment options for AMD are very limited, and include vitamin supplements and lifestyle changes. An exciting potential therapy currently being tested in clinical trials is transplantation of stem cell-derived RPE. Methods: We developed a NIH-registered embryonic stem line (CR-4), and in this study set out to determine if CR4-RPE are tolerated in normal mice and in murine models of retinal degeneration by injecting a bolus of CR4-RPE cells in the subretinal space of immunosuppressed wild-type, Mer mutant (Merkd), and Elovl4 deficient mice. Results: Mice with CR-RPE grafts were monitored daily, were examined routinely using OCT, and histology was prepared and examined at terminal end-points. Based on the parameters of the study, none of the animals with CR-RPE grafts (n=36) experienced any obvious adverse reactions. Conclusions: We conclude that transplanted CR-4 hES-derived RPE cells are well tolerated in immunosuppressed healthy and dystrophic murine retinas.

Low doses of BPA induced abnormal mitochondrial fission and hypertrophy in human embryonic stem cell-derived cardiomyocytes via the calcineurin-DRP1 signaling pathway: A comparison between XX and XY cardiomyocytes.

Humans are inevitably exposed to bisphenol A (BPA) via multiple exposure ways. Thus, attention should be raised to the possible adverse effects related to low doses of BPA. Epidemiological studies have outlined BPA exposure and the increased risk of cardiovascular diseases (such as cardiac hypertrophy), which has been confirmed to be sex-specific in rodent animals and present in few in vitro studies, although the molecular mechanism is still unclear. However, whether BPA at low doses equivalent to human internal exposure level could induce cardiac hypertrophy via the calcineurin-DRP1 signaling pathway by disrupting calcium homeostasis is unknown. To address this, human embryonic stem cell (H1, XY karyotype and H9, XX karyotype)-derived cardiomyocytes (CM) were purified and applied to study the low-dose effects of BPA on cardiomyocyte hypertrophy. In our study, when H1- and H9-CM were exposed to noncytotoxic BPA (8 ng/ml), markedly elevated hypertrophic-related mRNA expression levels (such as NPPA and NPPB), enhanced cellular area and reduced ATP supplementation, demonstrated the hypertrophic cardiomyocyte phenotype in vitro. The excessive fission produced by BPA was promoted by CnAß-mediated dephosphorylation of DRP1. At the molecular level, the increase in cytosolic Ca2+ levels by low doses of BPA could discriminate between H1- and H9-CM, which may suggest a potential sex-specific hypertrophic risk in cardiomyocytes in terms of abnormal mitochondrial fission and ATP production by impairing CnAß-DRP1 signaling. In CnAß-knockdown cardiomyocytes, these changes were highly presented in XX-karyotyped cells, rather than in XY-karyotyped cells.

Drug Discovery Platform Targeting M. tuberculosis with Human Embryonic Stem Cell-Derived Macrophages.

A major limitation in anti-tuberculosis drug screening is the lack of reliable and scalable models for homogeneous human primary macrophage cells of non-cancer origin. Here we report a modified protocol for generating homogeneous populations of macrophage-like cells from human embryonic stem cells. The induced macrophages, referred to as iMACs, presented similar transcriptomic profiles and characteristic immunological features of classical macrophages and were permissive to viral and bacterial infection, in particular Mycobacterium tuberculosis (Mtb). More importantly, iMAC production was amenable to scale up. To evaluate iMAC efficiency in high-throughput anti-tuberculosis drug screening, we performed a phenotypic screening against intracellular Mtb, involving a library of 3,716 compounds that included FDA-approved drugs and other bioactive compounds. Our primary screen identified 120 hits, which were validated in a secondary screen by dose-intracellular and -extracellular Mtb assays. Our confirmatory studies identified a novel anti-Mtb compound, 10-DEBC, also showing activity against drug-resistant strains.

Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells.

BACKGROUND: As the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming is not initiated in vitro. Previous studies found that vitamin C can enhance Tet (ten-eleven translocation) enzyme expression and improve 5hmC level in cells. But the effect of vitamin C supplementation on hPGC in vitro induction is still unknown. METHODS: We generated a gene-edited human embryonic stem cell (hESC) line carrying a BLIMP1-mkate2 reporter by CRISPR/Cas9 technology and used flow cytometry to optimize the PGC differentiation protocol; meanwhile, the expression of PGC genes (BLIMP1, TFAP2C, SOX17, OCT4) was evaluated by qRT-PCR. When different concentrations of vitamin C were added to the induction medium, the percentage of hPGCLCs (hPGC-like cells) was analyzed by flow cytometry; dot blot and ELISA were used to detect the levels of 5hmC and 5mC. The expression of TET enzymes was also evaluated by qRT-PCR. RESULTS: We optimized the PGC differentiation protocol with the BLIMP1-mkate reporter hESCs, and the efficiency of PGC induction in vitro can be improved to 30~40%. When 50 µg/mL vitamin C was added, the derived hPGCLCs not only upregulated the expression of key genes involved in human early germ cell development such as NANOS3, TFAP2C, BLIMP1, and SOX17, but also increased the levels of 5hmC and TET enzymes. CONCLUSIONS: Taken together, supplementation of vitamin C can promote the in vitro induction of hPGCLCs from hESCs, which might be related to vitamin C-mediated epigenetic regulations during the differentiation process.