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The gene of ATP-binding cassette subfamily C member 8 (Abcc8) is cytogenetically located at 11p15.1 and encodes the sulfonylurea receptor (SUR1). SUR1 is a subunit of ATP-sensitive potassium channel (KAPT) in the β-cell regulating insulin secretion. Mutations of ABCC8 are responsible for congenital hyperinsulinism (CHI). Here we reported that an Abcc8 heterozygous mutant cell line was generated by CRISPR/Cas9 technique with 1bp insertion resulting in abnormal splicing on human embryonic stem cell line H1. The phenotypic characteristics of this cell line reveal defective KATP channel and diazoxide-responsive that provides ideal model for molecular pathology research and drug screening for CHI. View Publication

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However,most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5),a known tumour suppressor and growth arrest-related lncRNA,is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal,but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis,we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore,we propose that the above pluripotency factors,GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific,our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms. View Publication

Bacterial magnetic particles (BacMPs) are efficient platforms of proteins for surface display systems. In this study,mononuclear cells from peripheral blood were separated using BacMPs expressing protein A on the BacMP membrane surface (protein A-BacMPs),which were complexed with the Fc fragment of anti-mouse IgG antibody. The procedure of positive selection involves incubation of mononuclear cells and mouse monoclonal antibodies against different cell surface antigens (CD8,CD14,CD19,CD20) prior to treatment with protein A-BacMP binding with rabbit anti-mouse IgG secondary antibodies. Flow cytometric analysis showed that approximately 97.5 +/- 1.7% of CD19(+) and CD20(+) cells were involved in the positive fraction after magnetic separation. The ratio of the negative cells in the negative fraction was approximately 97.6 +/-1.4%. This indicates that CD19(+) and CD20(+) cells can be efficiently separated from mononuclear cells. Stem cell marker (CD34) positive cells were also separated using protein A-BacMP binding with antibody. May-Grunwald Giemsa stain showed a high nuclear/cytoplasm ratio,which indicates a typical staining pattern of stem cells. The separated cells had the capability of colony formation as hematopoietic stem cells. Furthermore,the inhibitory effect of magnetic cell separation on CD14(+) cells was evaluated by measurement of cytokine in the culture supernatant by ELISA when the cells were cultured with or without lipopolysaccharide (LPS). The induction of IL1-beta,TNFalpha,and IL6 was observed in the presence of 1 ng/mL LPS in all fractions. On the other hand,in the absence of LPS,BacMPs had little immunopotentiation to CD14(+) cells as well as that of artificial magnetic particles,although TNFalpha and IL6 were slightly induced in the absence of LPS in the positive fraction. View Publication

Global gene expression profiling was performed on murine embryonic stem cells (ESCs) induced to differentiate by removal of leukemia inhibitory factor (LIF) to identify genes whose change in expression correlates with loss of pluripotency. To identify appropriate time points for the gene expression analysis,the dynamics of loss of pluripotency were investigated using three functional assays: chimeric mouse formation,embryoid body generation,and colony-forming ability. A rapid loss of pluripotency was detected within 24 hours,with very low residual activity in all assays by 72 hours. Gene expression profiles of undifferentiated ESCs and ESCs cultured for 18 and 72 hours in the absence of LIF were determined using the Affymetrix GeneChip U74v2. In total,473 genes were identified as significantly differentially expressed,with approximately one third having unknown biological function. Among the 275 genes whose expression decreased with ESC differentiation were several factors previously identified as important for,or markers of,ESC pluripotency,including Stat3,Rex1,Sox2,Gbx2,and Bmp4. A significant number of the decreased genes also overlap with previously published mouse and human ESC data. Furthermore,several membrane proteins were among the 48 decreased genes correlating most closely with the functional assays,including the stem cell factor receptor c-Kit. Through identification of genes whose expression closely follows functional properties of ESCs during early differentiation,this study lays the foundation for further elucidating the molecular mechanisms regulating the maintenance of ESC pluripotency and facilitates the identification of more reliable molecular markers of the undifferentiated state. View Publication

Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However,precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs,and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase,which blocks entry into mitosis by phosphorylating CDK1 at Y15,was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal,but not ectodermal,lineages. Functionally,WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775. View Publication

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived,patient-specific pluripotent cells for use in cell-based regenerative therapies. However,current methods of cell culture are tedious and expensive,and the mechanisms underlying cell proliferation are not understood. In this study,we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion,survival,and proliferation. We show that iPSC lines generated using Oct-3/4,Sox-2,Nanog,and Lin-28 express a repertoire of integrins similar to that of hESCs,with prominent expression of subunits alpha5,alpha6,alphav,beta1,and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin,in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel,as shown by immunological blockade experiments. On vitronectin,the integrin alphavbeta5 was required for initial attachment,but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore,iPSCs cultured on vitronectin for 9 passages retained normal karyotype,pluripotency marker expression,and capacity to differentiate in vitro. These studies suggest that vitronectin,or derivatives thereof,might substitute for Matrigel in a more defined system for iPSC culture. View Publication

OBJECTIVES: The goal of this study was to modify mesenchymal stem cells (MSCs) cells with a hypoxia-regulated heme oxygenase-1 (HO-1) plasmid to enhance the survival of MSCs in acute myocardial infarction (MI) heart. BACKGROUND: Although stem cells are being tested clinically for cardiac repair,graft cells die in the ischemic heart because of the effects of hypoxia/reoxygenation,inflammatory cytokines,and proapoptotic factors. Heme oxygenase-1 is a key component in inhibiting most of these factors. METHODS: Mesenchymal stem cells from bone marrow were transfected with either HO-1 or LacZ plasmids. Cell apoptosis was assayed in vitro after hypoxia-reoxygen treatment. In vivo,1 x 10(6) of male MSC(HO-1),MSC(LacZ),MSCs,or medium was injected into mouse hearts 1 h after MI (n = 16/group). Cell survival was assessed in a gender-mismatched transplantation model. Apoptosis,left ventricular remodeling,and cardiac function were tested in a gender-matched model. RESULTS: In the ischemic myocardium,the MSC(HO-1) group had greater expression of HO-1 and a 2-fold reduction in the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive cells compared with the MSC(LacZ) group. At seven days after implantation,the survival MSC(HO-1) was five-fold greater than the MSC(LacZ) group; MSC(HO-1) also attenuated left ventricular remodeling and enhanced the functional recovery of infarcted hearts two weeks after MI. CONCLUSIONS: A hypoxia-regulated HO-1 vector modification of MSCs enhances the tolerance of engrafted MSCs to hypoxia-reoxygen injury in vitro and improves their viability in ischemic hearts. This demonstration is the first showing that a physiologically inducible vector expressing of HO-1 genes improves the survival of stem cells in myocardial ischemia. View Publication

We have generated Tie2Cre+alpha4(f/f) mice with documented alpha4-integrin ablation in hematopoietic and endothelial cells. A prominent feature in this model is a sustained,significant increase in circulating progenitors at levels higher than the levels seen with Tie2Cre+VCAM-1(f/f) mice. To test whether phenotypic differences are due to contributions by ligands other than VCAM-1 in bone marrow,or to alpha4-deficient endothelial cells or pericytes,we carried out transplantation experiments using these mice as donors or as recipients. Changes in progenitor biodistribution after transplantation were seen only with alpha4-deficient donor cells,suggesting that these cells were necessary and sufficient to reproduce the phenotype with no discernible contribution by alpha4-deficient nonhematopoietic cells. Because several similarities are seen after transplantation between our results and those with CXCR4-/- donor cells,the data suggest that VLA4/VCAM-1 and CXCR4/CXCL12 pathways contribute to a nonredundant,ongoing signaling required for bone marrow retention of progenitor cells during homeostasis. View Publication

Human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs),a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS),could provide an unlimited source of cells for neural-related cell-based therapies and disease modeling. However,the use of NPCs for the study and treatment of a variety of debilitating neurological diseases requires the development of scalable and reproducible protocols for their generation,expansion,characterization,and neuronal differentiation. Here,we describe a serum-free method for the stepwise generation of NPCs from hPSCs through the sequential formation of embryoid bodies (EBs) and neuro-epithelial-like rosettes. NPCs isolated from neural rosette cultures can be homogenously expanded while maintaining high expression of pan-neural markers such as SOX1,SOX2,and Nestin. Finally,this protocol allows for the robust differentiation of NPCs into microtubule-associated protein 2 (MAP2) and β-Tubulin-III (β3T) positive neurons. View Publication

The complement activation product,C5a,is a pivotal member of the innate immune response; however,a diverse number of nonimmune functions are now being ascribed to C5a signaling,including roles during embryonic development. Here,we identify the expression of the C5a precursor protein,C5,as well as the C5a receptors,C5aR and C5L2,in both human embryonic stem cells and human-induced pluripotent stem cells. We show that administration of a physiologically relevant dose of purified human C5a (1 nM) stimulates activation of ERK1/2 and AKT signaling pathways,and is able to promote maintenance of the pluripotent state in the absence of FGF2. C5a also reduced cell loss following dissociation of human pluripotent stem cells. Our results reveal that complement C5a signaling supports human stem cell pluripotency and survival,and thus may play a key role in shaping early human embryonic development. Stem Cells 2014;32:3278-3284. View Publication