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The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes,and forced expression of OCT4,KLF4,and KLF2 allows maintenance of human cells in a naïve state [Hanna J,et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid,followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics,antibody labeling profile,gene expression,X-inactivation profile,mitochondrial morphology,microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive,but attainable,process,leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible. View Publication

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture,they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands,a process which we call vasculogenesis,was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum,fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response,which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis,the development of blood vessels from in situ differentiating endothelial cells,and angiogenesis,the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated. View Publication

OBJECTIVE The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However,the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression. METHODS The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ) in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR,immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR). Cell proliferation ability was detected by cell growth curve and MTT assay. RESULTS The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (Ptextless0.005). KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (Ptextless0.01) and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = -0.486,P = 0.003). Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza),the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased,the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased. CONCLUSION KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene's function as a tumor suppressor in cervical carcinogenesis. View Publication

Molecular markers defining self-renewing pluripotent embryonic stem cells (ESCs) have been identified by relative comparisons between undifferentiated and differentiated cells. Most of analysis has been done under a specific differentiation condition that may present significantly different molecular changes over others. Therefore,it is currently unclear if there are true consensus markers defining undifferentiated hESCs. To identify a set of key genes consistently altered during differentiation of hESCs regardless of differentiation conditions we have performed microarray analysis on undifferentiated hESCs (H1 and H9) and differentiated EB's and validated our results using publicly available expression array data sets. We constructed consensus modules by Weighted Gene Correlation Analysis (WGCNA) and discovered novel markers that are consistently present in undifferentiated hESCs under various differentiation conditions. We have validated top markers (downregulated: LCK,KLKB1 and SLC7A3; upregulated: RhoJ,Zeb2 and Adam12) upon differentiation. Functional validation analysis of LCK in self-renewal of hESCs by using LCK inhibitor or gene silencing with siLCK resulted in a loss of undifferentiation characteristics- morphological change,reduced alkaline phosphatase activity and pluripotency gene expression,demonstrating a potential functional role of LCK in self-renewal of hESCs. We have designated hESC markers to interactive networks in the genome,identifying possible interacting partners and showing how new markers relate to each other. Furthermore,comparison of these data sets with available datasets from iPSCs revealed that the level of these newly identified markers were correlated to the establishment of iPSCs,which may imply a potential role of these markers in gaining of cellular potency. Stem Cells 2014. View Publication

Charge-coupled device detectors are vulnerable to cosmic rays that can contaminate Raman spectra with positive going spikes. Because spikes can adversely affect spectral processing and data analyses,they must be removed. Although both hardware-based and software-based spike removal methods exist,they typically require parameter and threshold specification dependent on well-considered user input. Here,we present a fully automated spike removal algorithm that proceeds without requiring user input. It is minimally dependent on sample attributes,and those that are required (e.g.,standard deviation of spectral noise) can be determined with other fully automated procedures. At the core of the method is the identification and location of spikes with coincident second derivatives along both the spectral and spatiotemporal dimensions of two-dimensional datasets. The method can be applied to spectra that are relatively inhomogeneous because it provides fairly effective and selective targeting of spikes resulting in minimal distortion of spectra. Relatively effective spike removal obtained with full automation could provide substantial benefits to users where large numbers of spectra must be processed. View Publication

Background The AAVS1 locus is viewed as a ‘safe harbor' for transgene insertion into human genome. In the present study,we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected,stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting,25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette,we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Conclusions Given high targeting efficiency,stability in expression of inserted transgene and flexibility in transgene exchange,the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research,drug development,regenerative medicine and gene therapy. Copyright textcopyright 2013 John Wiley & Sons,Ltd. View Publication

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However,the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study,we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette,we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly,more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification,and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional,as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore,hESCs stably modified with the CD1d gene may serve as a convenient,unlimited,and competent DC source for iNKT cell-based cancer immunotherapy. View Publication

PURPOSE: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason,it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE.$$n$$nMETHODS: iPS-RPE was derived from human iPS cells. Immunocytochemistry,RT-PCR,and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT),RPE65,cellular retinaldehyde-binding protein (CRALBP),and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids.$$n$$nRESULTS: Cultured iPS-RPE expresses visual cycle genes LRAT,CRALBP,and RPE65. After incubation with all-trans retinol,iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly,after incubation with all-trans retinol,iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media.$$n$$nCONCLUSIONS: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally,the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE. View Publication

Asymmetry of cell fate is one fundamental property of stem cells,in which one daughter cell self-renews,whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms,such as plants,fungi,and mammals,has already been shown. However,before this current work,asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs),and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing,differentiating human and mouse ESCs. Moreover,we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3),indicating a molecular mechanism that regulates this phenomenon. Furthermore,our data support the hypothesis that retention of chromatids with the old" template DNA preserves the epigenetic memory of cell fate� View Publication

The fabrication of high-density polymer microarray is described,allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance. View Publication

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However,purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here,we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins,gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells,MLC-2v selected CMs had larger action potential amplitudes and durations. In addition,by immunofluorescence,we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However,only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach,it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. ?? 2013 Elsevier B.V. View Publication

Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population,and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular,disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors,aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits,we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as globulomers"� View Publication