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BACKGROUND This study aimed to evaluate the effect of exosomes produced by human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury. METHODS Exosomes were isolated and concentrated from conditioned medium using ultracentrifugation and ultrafiltration. hiPSC-MSCs-Exo were injected systemically via the inferior vena cava in a rat model of 70% warm hepatic I/R injury,and the therapeutic effect was evaluated. The serum levels of transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were measured using an automatic analyzer. The expression of inflammatory factors was measured using enzyme-linked immunosorbent assay (ELISA). Histological changes indicated changes in pathology and inflammatory infiltration in liver tissue. Apoptosis of hepatic cells in liver tissue was measured using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining along with apoptotic markers. RESULTS hiPSCs were efficiently induced into hiPSC-MSCs with typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 50 to 60 nm and expressed exosomal markers (CD9,CD63 and CD81). Hepatocyte necrosis and sinusoidal congestion were markedly suppressed with a lower Suzuki score after hiPSC-MSCs-Exo administration. The levels of the hepatocyte injury markers AST and ALT were significantly lower in the treated group than in the control group. Inflammatory markers,such as tumor necrosis factor (TNF)-α,interleukin (IL)-6 and high mobility group box 1 (HMGB1),were significantly reduced after administration of hiPSC-MSCs-Exo,which suggests that the exosomes have a role in suppressing the inflammatory response. Additionally,in liver tissues from the experimental group,the levels of apoptotic markers,such as caspase-3 and bax,were significantly lower and the levels of oxidative markers,such as glutathione (GSH),glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD),were significantly higher than in the control group. These data point to an anti-apoptotic,anti-oxidative stress response role for hiPSC-MSCs-Exo. CONCLUSIONS Our results demonstrated that hiPSC-MSCs-Exo alleviate hepatic I/R injury,possibly via suppression of inflammatory responses,attenuation of the oxidative stress response and inhibition of apoptosis. View Publication

Understanding how memory B cells are induced and relate to long-lived plasma cells is important for vaccine development. Immunity to oral vaccines has been considered short-lived because of a poor ability to develop IgA B-cell memory. Here we demonstrate that long-lived mucosal IgA memory is readily achieved by oral but not systemic immunization in mouse models with NP hapten conjugated with cholera toxin and transfer of B1-8(high)/GFP(+) NP-specific B cells. Unexpectedly,memory B cells are poorly related to long-lived plasma cells and less affinity-matured. They are α4β7-integrin(+)CD73(+)PD-L2(+)CD80(+) and at systemic sites mostly IgM(+),while 80% are IgA(+) in Peyer's patches. On reactivation,most memory B cells in Peyer's patches are GL7(-),but expand in germinal centres and acquire higher affinity and more mutations,demonstrating strong clonal selection. CCR9 expression is found only in Peyer's patches and appears critical for gut homing. Thus,gut mucosal memory possesses unique features not seen after systemic immunization. View Publication

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and,thereby,leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies,IGHV1-2(∗)02-rearranging mice,which also express a VRC01-antibody precursor light chain,can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages. View Publication

G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth,death,movement,transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study,we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol,we found that the expression of several chemokine receptors changed dramatically during the hESC/NSC transition. Especially,the expression of CXCR4 increased approximately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted,whereas the antagonist AMD3100 delayed the neural induction process. In consistence with antagonizing CXCR4,knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together,our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine. View Publication

The mechanisms of how signaling pathways are coordinated and integrated for the maintenance of the self-renewal of human embryonic stem cells (hESCs) and the acquisition of pluripotency in reprogramming are still only partly understood. CDK1 is a key regulator of mitosis. Recently,CDK1 has been shown to be involved in regulating self-renewal of stem cells,even though the mechanistic role of how CDK1 regulates pluripotency is unknown. In this report,we aim to understand how CDK1 can control pluripotency by reducing CDK1 activity to a level that has no effect on cell cycle progression. We demonstrated that high levels of CDK1 is associated with the pluripotency stage of hESCs; and decreased CDK1 activity to a level without perturbing the cell cycle is sufficient to induce differentiation. CDK1 specifically targets the phosphorylation of PDK1 and consequently the activity of PI3K/Akt and its effectors ERK and GSK3β. Evidence of the reversion of inactive CDK1-mediated differentiation by the inhibition of Akt signaling effectors suggests that the CDK1-PDK1-PI3K/Akt kinase cascade is a functional signaling pathway for the pluripotency of hESCs. Moreover,cyclin B1-CDK1 complexes promote somatic reprogramming efficiency,probably by regulating the maturation of induced pluripotent stem cells (iPSCs),as cyclin B1 stimulates a higher cellular level of LIN28A,suggesting that monitoring iPSC factors could be a new path for the enhancement of reprogramming efficiency. Together,we demonstrate an essential role for the CDK1-PDK1-PI3K/Akt kinase signaling pathway in the regulation of self-renewal,differentiation,and somatic reprogramming,which provides a novel kinase cascade mechanism for pluripotency control and acquisition.Cell Death and Differentiation advance online publication,16 September 2016; doi:10.1038/cdd.2016.84. View Publication

Wnt signaling is a key regulator of vertebrate heart development; however,specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components,monitor pathway activity,and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A,via FZD7 and canonical signaling,regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B,via ROR2 and noncanonical signaling,regulate MESP1 expression and cardiovascular development; and that later in development WNT2,WNT5A/5B,and WNT11,via FZD4 and FZD6,regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis,and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development. View Publication

Mitochondria are critical to neurogenesis,but the mechanisms of mitochondria in neurogenesis have not been well explored. We fully characterized mitochondrial alterations and function in relation to the development of human induced pluripotent stem cell (hiPSC)-derived dopaminergic (DA) neurons. Following directed differentiation of hiPSCs to DA neurons,mitochondria in these neurons exhibit pronounced changes during differentiation,including mature neurophysiology characterization and functional synaptic network formation. Inhibition of mitochondrial respiratory chains via application of complex IV inhibitor KCN (potassium cyanide) or complex I inhibitor rotenone restricted neurogenesis of DA neurons. These results demonstrated the direct importance of mitochondrial development and bioenergetics in DA neuronal differentiation. Our study also provides a neurophysiologic model of mitochondrial involvement in neurogenesis,which will enhance our understanding of the role of mitochondrial dysfunctions in neurodegenerative diseases. View Publication

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs),but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs,sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study,we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK,ATP in single hESC-VCMs was dormant under baseline conditions,but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ˜8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly,sarcIK,ATP displayed a ˜3-fold increase after treatment with hypoxia (5% O2). MitoIK,ATP was absent in hESC-VCMs. However,the thyroid hormone T3 up-regulated mitoIK,ATP,conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system,T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK,ATP. View Publication

Wnt signaling plays essential roles in both embryonic pattern formation and postembryonic tissue homoestasis. High levels of Wnt activity repress foregut identity and facilitate hindgut fate through forming a gradient of Wnt signaling activity along the anterior-posterior axis. Here,we examined the mechanisms of Wnt signaling in hindgut development by differentiating human embryonic stem cells (hESCs) into the hindgut progenitors. We observed severe morphological changes when Wnt signaling was blocked by using Wnt antagonist Dkk1. We performed deep-transcriptome sequencing (RNA-seq) and identified 240 Wnt-activated genes and 2023 Wnt-repressed genes,respectively. Clusters of Wnt targets showed enrichment in specific biological functions,such as gastrointestinal or skeletal development" in the Wnt-activated targets and "neural or immune system development" in the Wnt-repressed targets. Moreover� View Publication

The sodium bicarbonate co-transporter (NBC) is the major bicarbonate-dependent acid-base transporter in mammalian astrocytes and has been implicated in ischemic brain injury. A malfunction of astrocytes could have great impact on the outcome of stroke due to their participation in the formation of blood-brain barrier,synaptic transmission,and electrolyte balance in the human brain. Nevertheless,the role of NBC in the ischemic astrocyte death has not been well understood. In this work,we obtained skin biopsies from healthy human subjects and had their fibroblasts grown in culture and reprogrammed into human-induced pluripotent stem cells (hiPSCs). These hiPSCs were further differentiated into neuroprogenitor cells (NPCs) and then into human astrocytes. These astrocytes express GFAP and S100β and readily propagate calcium waves upon mechanical stimulation. Using pH-sensitive dye BCECF [2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein] and qPCR technique,we have confirmed that these astrocytes express functional NBC including electrogenic NBC (NBCe). In addition,astrocytes exposed to an ischemic solution (IS) that mimics the ischemic penumbral environment enhanced both mRNA and protein expression level of NBCe1 in astrocytes. Using IS and a generic NBC blocker S0859,we have studied the involvement of NBC in IS-induced human astrocytes death. Our results show that a 30μM S0859 induced a 97.5±1.6% (n=10) cell death in IS-treated astrocytes,which is significantly higher than 43.6±4.5%,(n=10) in the control group treated with IS alone. In summary,a NBC blocker exaggerates IS-induced cell death,suggesting that NBC activity is essential for astrocyte survival when exposed to ischemic penumbral environment. View Publication

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs),and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the $\beta$-globin gene (HBB). Sickle hemoglobin damages erythrocytes,causing vasoocclusion,severe pain,progressive organ damage,and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA,together with a single-stranded DNA oligonucleotide donor (ssODN),to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice,ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing,enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells,and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases. View Publication

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells,methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here,we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped,submillimeter colonies. After 42 h of BMP4 stimulation,cells form self-organized differentiation patterns in concentric radial domains,which express specific markers associated with the embryonic germ layers,reminiscent of gastrulating embryos. Our protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO),human laminin-521 (LN-521) as extracellular matrix coating,and either conditioned or chemically defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. View Publication