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Manipulation of genes in human embryonic stem cells (hESCs) is imperative for their highly potential applications; however,the transduction efficiency remains very low. Although existing evidence revealed the type,size,and zeta potential of vector affect gene transfection efficiency in cells,the systematic study in hESCs is scarce. In this study,using poly(amidoamine) (PAMAM) dendrimers ended with amine,hydroxyl,or carboxyl as model,we tested the influences of size and surface group as well as cytotoxicity and endocytosis on hESC gene transfection. We found that in culture medium of mTeSR the particle sizes of G5,G7,G4.5COOH,and G5OH were around 5 nm and G1 had a smaller size of 3.14 nm. G5 and G7 had a slight and significant positive zeta potential,respectively,whereas G1 was slightly negative,and G4.5COOH and G5OH were significantly negative. We demonstrated that only amine-terminated dendrimers accomplished gene transfection in hESCs,which is greater than that from Lipofectamine 2000 transfection. Ten micromolar G5 had the greatest efficiency and was better than 1000 μM G1. Only a low concentration (0.5 and 1 μM) of G7 realized gene delivery. Amine-ended dendrimers,especially with higher generations,were detrimental to the growth and pluripotent maintenance of hESCs. In contrast,similarly sized hydroxyl- and carboxyl-terminated dendrimers exerted much lower cytotoxicity,in which carboxyl-terminated dendrimer maintained pluripotency of hESCs. We also confirmed the endocytosis into and significant exocytosis from hESCs using FITC-labeled G5 dendrimer. These results suggested that careful considerations of size,concentration,and zeta potential,particularly the identity and position of groups,as well as minimized exocytosis in the design of a vector for hESC gene delivery are necessary,which helps to better design an effective vector in hESC gene transduction. View Publication

Functional hepatocytes derived from human pluripotent stem cells (hPSCs) have potential as tools for predicting drug-induced hepatotoxicity in the early phases of drug development. However,the propensity of hPSC lines to differentiate into specific lineages is reported to differ. The ability to predict low propensity of hPSCs to differentiate into hepatocytes would facilitate the selection of useful hPSC clones and substantially accelerate development of hPSC-derived hepatocytes for pharmaceutical research. In this study,we compared the expression of genes associated with hepatic differentiation in five hPSC lines including human ES cell line,H9,which is known to differentiate into hepatocytes,and an hPSC line reported with a poor propensity for hepatic differentiation. Genes distinguishing between undifferentiated hPSCs,hPSC-derived hepatoblast-like differentiated cells,and primary human hepatocytes were drawn by two-way cluster analysis. The order of expression levels of genes in undifferentiated hPSCs was compared with that in hPSC-derived hepatoblast-like cells. Three genes were selected as predictors of low propensity for hepatic differentiation. Expression of these genes was investigated in 23 hPSC clones. Review of representative cells by induction of hepatic differentiation suggested that low prediction scores were linked with low hepatic differentiation. Thus,our model using gene expression ranking and bioinformatic analysis could reasonably predict poor differentiation propensity of hPSC lines. View Publication

Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs),dental pulp stem cells (hDPSCs) and bone marrow MSCs (hBMSCs) are exciting cell sources in regenerative medicine. However,there has been no report comparing hDPSCs,hBMSCs and hiPSC-MSCs for bone engineering in an injectable calcium phosphate cement (CPC) scaffold. The objectives of this study were to: (1) develop a novel injectable CPC containing hydrogel fibers encapsulating stem cells for bone engineering,and (2) compare cell viability,proliferation and osteogenic differentiation of hDPSCs,hiPSC-MSCs from bone marrow (BM-hiPSC-MSCs) and from foreskin (FS-hiPSC-MSCs),and hBMSCs in CPC for the first time. The results showed that the injection did not harm cell viability. The porosity of injectable CPC was 62%. All four types of cells proliferated and differentiated down the osteogenic lineage inside hydrogel fibers in CPC. hDPSCs,BM-hiPSC-MSCs,and hBMSCs exhibited high alkaline phosphatase,runt-related transcription factor,collagen I,and osteocalcin gene expressions. Cell-synthesized minerals increased with time (ptextless0.05),with no significant difference among hDPSCs,BM-hiPSC-MSCs and hBMSCs (ptextgreater0.1). Mineralization by hDPSCs,BM-hiPSC-MSCs,and hBMSCs inside CPC at 14d was 14-fold that at 1d. FS-hiPSC-MSCs were inferior in osteogenic differentiation compared to the other cells. In conclusion,hDPSCs,BM-hiPSC-MSCs and hBMSCs are similarly and highly promising for bone tissue engineering; however,FS-hiPSC-MSCs were relatively inferior in osteogenesis. The novel injectable CPC with cell-encapsulating hydrogel fibers may enhance bone regeneration in dental,craniofacial and orthopedic applications. View Publication

Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular,hematological,and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT(+) and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2,OCT4,KLF-4,and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72 h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2(-/-),$\$-chain(-/-),C2(-/-) immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment,immunostaining,and RT-PCR. Additionally,the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery. View Publication

Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM),umbilical cord blood (UCB),and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study,we have identified ALDH(+) cells within human skeletal muscles,and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH,Aldefluor,identified brightly stained (ALDH(br)) cells with low side scatter (SSC(lo)),in enzymatically dissociated muscle biopsies,thereafter abbreviated as SMALD(+) (for skeletal muscle ALDH(+)) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD(+)/CD34(-) and SMALD(+)/CD34(+) cells. These sub-populations did not initially express endothelial (CD31),hematopoietic (CD45),and myogenic (CD56) markers. Upon sorting,however,whereas SMALD(+)/CD34(+) cells developed in vitro as a heterogeneous population of CD56(-) cells able to differentiate in adipoblasts,the SMALD(+)/CD34(-) fraction developed in vitro as a highly enriched population of CD56(+) myoblasts able to form myotubes. Moreover,only the SMALD(+)/CD34(-) population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. View Publication

Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore,we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics,BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm),yet,24 hr later,suppressed endoderm and induced mesoderm. At lineage bifurcations,cross-repressive signals separated mutually exclusive fates; TGF-?? and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations,thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of pre-enhancer" states before activation� View Publication

Incurable neurological disorders such as Parkinson's disease (PD),Huntington's disease (HD),and Alzheimer's disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases,we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor,valproic acid (VPA),and inhibitor of p160-Rho associated coiled-coil kinase (ROCK),Y-27632,after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology,cell surface antigens,pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542,inhibitors of the SMAD signaling pathway,HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction,neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture,which had the ability to differentiate further into NPCs and neurons,as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays. View Publication

BACKGROUND: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.backslashnbackslashnFINDINGS: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.backslashnbackslashnCONCLUSIONS: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic. View Publication

Aging is known to be the single most important risk factor for multiple diseases. Sirtuin 6,or SIRT6,has recently been identified as a critical regulator of transcription,genome stability,telomere integrity,DNA repair,and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging,we demonstrated that human dermal fibroblasts (HDFs) from older human subjects were more resistant to reprogramming by classic Yamanaka factors than those from younger human subjects,but the addition of SIRT6 during reprogramming improved such efficiency in older HDFs substantially. Despite the importance of SIRT6,little is known about the molecular mechanism of its regulation. We show,for the first,time posttranscriptional regulation of SIRT6 by miR-766 and inverse correlation in the expression of this microRNA in HDFs from different age groups. Our results suggest that SIRT6 regulates miR-766 transcription via a feedback regulatory loop,which has implications for the modulation of SIRT6 expression in reprogramming of aging cells. View Publication

Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development,disease modeling,drug discovery,and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined,growth factor- and serum-free system by temporal modulation of Wnt/$$-catenin signaling via small molecules. We demonstrate a 10-day,two-stage process that recapitulates endothelial cell development,in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described. View Publication