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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

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation,whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans,or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs,suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci,characteristic of the inactive X. Moreover,analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs. View Publication

Transcription factors NANOG,OCT4,and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however,their expression profiles during early differentiation of hES cells are unclear. In this study,we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG,OCT4,and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells,characterized by specific expression patterns of NANOG,OCT4,and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time,indicating a gradual mode of developmental transition in these cells. Notably,distinct regulation of SOX2 during early differentiation events was detected,highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation. View Publication

Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival,self-renewal,and differentiation. Thus,hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture. Soluble angiopoeitin-1 (Ang-1)-derived peptide QHREDGS added to defined serum-free media increased hPSC colony cell number and size during long- and short-term culture when grown on feeder cell layers or Matrigel,i.e. on standard substrates,without affecting hPSC morphology,growth rate or the ability to differentiate into multiple lineages both invitro and invivo. Importantly,QHREDGS treatment decreased hPSC apoptosis during routine passaging and single-cell dissociation. Mechanistically,the interaction of QHREDGS with ??1-integrins increased expression of integrin-linked kinase (ILK),increased expression and activation of extracellular signal-regulated kinases 1/2 (ERK1/2),and decreased caspase-3/7 activity. QHREDGS immobilization to polyethylene glycol hydrogels significantly increased cell adhesion in a dose-dependent manner. We propose QHREDGS as a small molecule inhibitor of hPSC apoptosis and the basis of an affordable defined substrate for hPSC maintenance. ?? 2014 Elsevier Ltd. View Publication

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells,up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking,however,decreased to 0.1% or lower within 2 weeks. In contrast,EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis,and EGFP expression was observed in CD4(+),CD8(+),CD20(+),and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452) View Publication

Embryonic stem cells (ESCs) exhibit unrestricted and indefinite,but stringently controlled,proliferation,and can differentiate into any lineage in the body. In the current study,we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs,we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines,a few RPGs also exhibit higher expression levels. In situ nuclear run-on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome-wide chromatin immunoprecipitation-deep sequencing and bioinformatics approaches,we discovered that,RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1,M,and G2 phases of the cell cycle. Interestingly,the rDNA repeats are marked by the activating H3K4me3 only in the M phase,and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc,a known regulator of cell growth and proliferation,occupies both the rRNA genes and RPGs. Functionally,down-regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together,our results show that expression of rRNA,which is regulated by the Myc pluripotency transcription factor,and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007-2013,2016. textcopyright 2016 Wiley Periodicals,Inc. View Publication

PURPOSE To determine the safety,survival,and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. METHODS Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN),group 2 (n = 59) received rCPCB-RPE1 implants,and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery),the eyes were examined histologically for cell survival,phagocytosis,and local toxicity. RESULTS Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry,suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. CONCLUSIONS These results demonstrate the safety,survival,and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model. View Publication

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology,including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore,in the current study,we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM),as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes,NQO1 and SRXN1. Interestingly,exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover,rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH(+)) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. View Publication

Long noncoding RNAs (lncRNAs) exhibit diverse functions,including regulation of development. Here,we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition,we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans,and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm. View Publication

The ability of human embryonic stem cells (hESCs) to proliferate indefinitely is attributed to its high telomerase activity and associated long telomere. However,factors regulating telomere length in hESCs remain largely uncharacterized. The aims of this study were,to identify factors which modulate telomere length of hESCs,and to determine if the telomere length of hESCs influences cellular senescence of its differentiated progeny cells. Telomerase reverse transcriptase (TERT) gene expression,telomerase activity and telomere length of hESCs cultured in different culture systems were compared. Genetically identical hESCs of different telomere lengths were differentiated into fibroblasts simultaneously,and the population doubling and cellular senescence levels were determined. We found that telomere lengths were significantly different in different culture systems and Fibroblast growth factor-2 (FGF-2) upregulated TERT expression,telomerase activity and telomere length via Wnt/β-catenin signaling pathway in hESCs in a significant manner. We also provide evidence that fibroblast differentiated from hESCs with longer telomere exhibited significant more population doublings and longer life span than those derived from hESCs with shorter telomeres. Thus,FGF-2 levels in hESCs culture systems can be manipulated to generate cells with longer telomere which would be advantageous in the applications of hESCs in regenerative medicine. View Publication

BACKGROUND: Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations,the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally,a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor,Y-27632,previously has been identified as enhancing survival of hESCs upon single-cell dissociation,as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: HESCs were sorted using markers for SSEA-3,TRA-1-81,and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions,cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically,treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632,hESCs were further analyzed. Specifically,hESCs sorted with and without the addition of Y-27632 retained normal morphology,expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry,and maintained a stable karyotype. In addition,the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. CONCLUSIONS/SIGNIFICANCE: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency,and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types,identification and isolation of stem cell subpopulations,and generation of single cell clones. Finally,these results demonstrate an additional application of ROCK inhibition to hESC research. View Publication