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CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which,however,has focused on HDR-based strategies and was proven inefficient. Here,we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs,and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy,integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells,and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. View Publication

Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells,however,the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase,a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues. View Publication

Genetic comparison between human embryonic stem cells and induced pluripotent stem cells has been hampered by genetic variation. To solve this problem,we have developed an isogenic system that allows direct comparison of induced pluripotent stem cells (hiPSCs) to their genetically matched human embryonic stem cells (hESCs). We show that hiPSCs have a highly similar transcriptome to hESCs. Global transcriptional profiling identified 102-154 genes (textgreater2 fold) that showed a difference between isogenic hiPSCs and hESCs. A stringent analysis identified NNAT as a key imprinted gene that was dysregulated in hiPSCs. Furthermore,a disproportionate number of X-chromosome localized genes were over-expressed in female hiPSCs. Our results indicate that despite a remarkably close transcriptome to hESCs,isogenic hiPSCs have alterations in imprinting and regulation of X-chromosome genes. View Publication

Parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells. Although previous studies have led to the theory that the basis of this tropism is receptor expression,this has been questioned by more recent observation. In the study reported here,we have investigated the basis of this tropism,and a potential role of erythropoietin (Epo) signaling,in erythroid progenitor cells (EPCs) expanded ex vivo from CD34(+) hematopoietic cells in the absence of Epo (CD36(+)/Epo(-) EPCs). We show,first,that CD36(+)/Epo(-) EPCs do not support B19V replication,in spite of B19V entry,but Epo exposure either prior to infection or after virus entry enabled active B19V replication. Second,when Janus kinase 2 (Jak2) phosphorylation was inhibited using the inhibitor AG490,phosphorylation of the Epo receptor (EpoR) was also inhibited,and B19V replication in ex vivo-expanded erythroid progenitor cells exposed to Epo (CD36(+)/Epo(+) EPCs) was abolished. Third,expression of constitutively active EpoR in CD36(+)/Epo(-) EPCs led to efficient B19V replication. Finally,B19V replication in CD36(+)/Epo(+) EPCs required Epo,and the replication response was dose dependent. Our findings demonstrate that EpoR signaling is absolutely required for B19V replication in ex vivo-expanded erythroid progenitor cells after initial virus entry and at least partly accounts for the remarkable tropism of B19V infection for human erythroid progenitors. View Publication

SIRT1,an NAD-dependent deacetylase,plays a role in regulation of autophagy. SIRT1 increases mitochondrial function and reduces oxidative stress,and has been linked to age-related reactive oxygen species (ROS) generation,which is highly dependent on mitochondrial metabolism. H2O2 induces oxidative stress and autophagic cell death through interference with Beclin 1 and the mTOR signaling pathways. We evaluated connections between SIRT1 activity and induction of autophagy in murine (m) and human (h) embryonic stem cells (ESCs) upon ROS challenge. Exogenous H2O2 (1 mM) induced apoptosis and autophagy in wild-type (WT) and Sirt1-/- mESCs. High concentrations of H2O2 (1 mM) induced more apoptosis in Sirt1-/-,than in WT mESCs. However,addition of 3-methyladenine,a widely used autophagy inhibitor,in combination with H2O2 induced more cell death in WT than in Sirt1-/- mESCs. Decreased induction of autophagy in Sirt1-/- mESCs was demonstrated by decreased conversion of LC3-I to LC3-II,lowered expression of Beclin-1,and decreased LC3 punctae and LysoTracker staining. H2O2 induced autophagy with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics in Sirt1-/- mESCs. Increased phosphorylation of P70/85-S6 kinase and ribosomal S6 was noted in Sirt1-/- mESCs,suggesting that SIRT1 regulates the mTOR pathway. Consistent with effects in mESCs,inhibition of SIRT1 using Lentivirus-mediated SIRT1 shRNA in hESCs demonstrated that knockdown of SIRT1 decreased H2O2-induced autophagy. This suggests a role for SIRT1 in regulating autophagy and mitochondria function in ESCs upon oxidative stress,effects mediated at least in part by the class III PI3K/Beclin 1 and mTOR pathways. Stem Cells 2014;32:1183-1194 View Publication

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report,human mesenchymal cells (hMSCs) generated from the human ES cell line H9,were reprogrammed back to induced pluripotent state using Oct-4,Sox2,Nanog,and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes,lipid droplets,glycogen,scarce reticulum) and nuclear levels (features of nuclear plasticity,presence of euchromatin,reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal-epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. View Publication

Recent studies have suggested that REX1 (reduced expression 1) plays an important role in pluripotency,proliferation,and differentiation. However,the molecular mechanisms involved in REX1-dependent regulation of diverse cellular processes remain unclear. To elucidate the regulatory functions of REX1 in human embryonic stem cells (hESCs),comparative proteomic analysis was performed on REX1 RNAi specifically silenced hESCs. Analysis of the proteome via nano-LC-MS/MS identified 140 differentially expressed proteins (DEPs) displaying a textgreater2-fold difference in expression level between control and REX1 knockdown (KD) hESCs,which were then compared with transcriptome data and validated by quantitative real-time RT-PCR and Western blotting. These DEPs were analyzed by GO,pathway,and functional clustering analyses to determine the molecular functions of the proteins and pathways regulated by REX1. The REX1 KD-mediated DEPs mapped to major biological processes involved in the regulation of ribosome-mediated translation and mitochondrial function. Functional network analysis revealed a highly interconnected network among these DEPs and indicated that these interconnected proteins are predominantly involved in translation and the regulation of mitochondrial organization. These findings regarding REX1-mediated regulatory network have revealed the contributions of REX1 to maintaining the status of hESCs and have improved our understanding of the molecular events that underlie the fundamental properties of hESCs. View Publication

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) $$$$ and $$$$ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells. View Publication

Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs),including human embryonic stem cells and induced pluripotent stem cells,is a complex,challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study,we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells,which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion,our study demonstrates that the HepaRG ACM,a hepatic progenitor-specific matrix,plays an important role in the hepatic differentiation of hPSCs. View Publication

Autologous bone grafting represents the gold standard modality to treat atrophic non-unions by virtue of its osteoinductive and osteoconductive properties. The common harvest site is the iliac crest,but there are major concerns due to limited volume and considerable donor site morbidity. Alternative autologous bone graft can be harvested from the femoral bone cavity using a newly developed 'Reamer Irrigator Aspirator' (RIA). Osseous aspirated particles can be recovered with a filter and used as auto-graft. The purpose of this study was to compare the concentration and differentiation potential of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) harvested with the RIA technique or from the iliac crest,respectively. RIA aspirate was collected from 26 patients undergoing intramedullary nailing of femur fractures. Iliac crest aspirate was collected from 38 patients undergoing bone graft transplantation. Concentration of MSC and EPC were assessed by means of the MSC colony assay,EPC culture assay and flowcytometry (CD34,CD133,VEGF-R2),respectively. Osteogenic differentiation of MSC's was measured by von Kossa staining. Patients in both groups did not significantly differ regarding their age,gender or pre-existing health conditions. In comparison to aspirates obtained from iliac crest the RIA aspirates from the femur contained a significantly higher percentage of CD34+ progenitor cells,a significantly higher concentration of MSC and a significantly higher concentration of early EPC. The percentage of late EPC did not differ between both sites. Moreover,the capability of MSC for calcium deposition was significantly enhanced in MSC obtained with RIA. Our results show that RIA aspirate is a rich source for different types of autologous progenitor cells,which can be used to accelerate healing of bone and other musculoskeletal tissues. View Publication

Genome stability of human embryonic stem cells (hESC) is an important issue because even minor genetic alterations can negatively impact cell functionality and safety. The incorrect repair of DNA double-stranded breaks (DSBs) is the ultimate cause of the formation of chromosomal aberrations. Using G2 radiosensitivity assay,we analyzed chromosomal aberrations in pluripotent stem cells and somatic cells. The chromatid exchange aberration rates in hESCs increased manifold 2 hours after irradiation as compared with their differentiated derivatives,but the frequency of radiation-induced chromatid breaks was similar. The rate of radiation-induced chromatid exchanges in hESCs and differentiated cells exhibited a quadratic dose response,revealing two-hit mechanism of exchange formation suggesting that a non-homologous end joining (NHEJ) repair may contribute to their formation. Inhibition of DNA-PK,a key NHEJ component,by NU7026 resulted in a significant decrease in radiation-induced chromatid exchanges in hESCs but not in somatic cells. In contrast,NU7026 treatment increased the frequency of radiation-induced breaks to a similar extent in pluripotent and somatic cells. Thus,DNA-PK dependent NHEJ efficiently participates in the elimination of radiation-induced chromatid breaks during the late G2 in both cell types and DNA-PK activity leads to a high level of misrejoining specifically in pluripotent cells. View Publication