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Human cell transformation is a key step for oncogenic development,which involves multiple pathways; however,the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC),we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung,breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and,OCT4,in turn,stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover,we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a,promotes p63 but suppresses p53 expression,which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and,most likely,also to the iPSC process. View Publication

UNLABELLED Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development,as well as the state of differentiation of susceptible cells at the time of infection,affects the severity of the disease. We used human embryonic stem (ES) cell-derived primitive prerosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs,infection of ES cell-derived pNSCs with HCMV was nonprogressive. At a low multiplicity of infection,we observed only a small percentage of cells expressing immediate-early genes (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1,and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell associated and were mostly non-neural tropic. Finally,we found that viral genomes could persist in pNSC cultures for up to a month after infection despite the absence of detectable IE expression by immunofluorescence,and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion,our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. IMPORTANCE Human cytomegalovirus (HCMV) is a betaherpesvirus that is highly prevalent in the population. HCMV infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal HCMV disease range from deafness and learning disabilities to more severe symptoms such as microcephaly. In this study,we have used embryonic stem cells to generate primitive neural stem cells and have used these to model HCMV infection of the fetal central nervous system (CNS) in vitro. Our results reveal that these cells,which are similar to those present in the developing neural tube,do not support viral replication but instead likely constitute a viral reservoir. Future work will define the effect of viral persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral replication in the CNS. 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 reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly,we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4,SOX2,KLF4,and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology,immunocytochemistry,and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives,including neural stem cells,beating cardiomyocytes,and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications. View Publication

Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear,however,whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells,among which is E2F2,a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1,in addition to proliferation of hESC,indicated by a higher proportion of cells in G1,fewer cells in G2/M phase,and a reduced capacity to generate hESC colonies in vitro. Nonetheless,E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly,E2F2 knockdown in hESC significantly inhibited tumor growth in vivo,which was considerably smaller than tumors generated from control hESC,although displaying typical teratoma traits,a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness,providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells. View Publication

The breakthrough development of induced pluripotent stem cells (iPSCs) raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells. However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear. In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with autogenous peripheral blood mononuclear cells (PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation. However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs. Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells (CD3+CD8− T cells,CD3+CD8+ T cells or CD3−CD56+ NK cells) by NPCs in both PBMC and T cell co-culture systems. These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs. View Publication

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here,iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4,and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry,RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery. ?? 2013. View Publication

Ring chromosomes are structural aberrations commonly associated with birth defects,mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome,and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes,no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division,ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations,enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of /`chromosome therapy/' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition,our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control,which is of critical relevance to human development and disease. 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

Background Proteoglycans are found on the cell surface and in the extracellular matrix,and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate,and therefore represent an excellent tool to manipulate these pathways. Despite their importance,there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Methods Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages,and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis,immunoblotting,immunofluorescence and disaccharide analysis. Results Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1,HS6ST2 and HS6ST3,three heparan sulfate biosynthetic enzymes,within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Conclusions Differentiation of embryonic stem cells markedly changes the proteoglycanome. General significance The glycosaminoglycan biosynthetic pathway is complex and highly regulated,and therefore,understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. ?? 2014 Elsevier B.V. All rights reserved. View Publication

A defined xeno-free system for patient-specific iPSC derivation and differentiation is required for translation to clinical applications. However,standard somatic cell reprogramming protocols rely on using MEFs and xenogeneic medium,imposing a significant obstacle to clinical translation. Here,we describe a well-defined culture system based on xeno-free media and LN521 substrate which supported i) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages into hiPSCs with a 15-30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisable polycistronic vector and optimized culture conditions,we achieved up to 0.15%-0.3% reprogramming efficiencies. Subsequently,transgene-free hiPSCs were obtained by Cre-mediated excision of the reprogramming factors. The derived iPSCs maintained long-term self-renewal,normal karyotype and pluripotency,as demonstrated by the expression of stem cell markers and ability to form derivatives of three germ layers both in vitro and in vivo. Importantly,we demonstrated that Parkinson's patient transgene-free iPSCs derived using the same system could be directed towards differentiation into dopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robust platform for the generation of patient-specific iPSCs and derivatives for clinical and translational applications. textcopyright 2013 Elsevier Ltd. View Publication

There is a great deal of uncertainty on how low (≤ 0.1 Gy) doses of ionizing radiation (IR) affect human cells,partly due to a lack of suitable experimental model systems for such studies. The uncertainties arising from low-dose IR human data undermine practical societal needs to predict health risks emerging from diagnostic medical tests' radiation,natural background radiation,and environmental radiological accidents. To eliminate a variability associated with remarkable differences in radioresponses of hundreds of differentiated cell types,we established a novel,human embryonic stem cell (hESC)-based model to examine the radiobiological effects in human cells. Our aim is to comprehensively elucidate the gene expression changes in a panel of various hESC lines following low IR doses of 0.01; 0.05; 0.1 Gy; and,as a reference,relatively high dose of 1 Gy of IR. Here,we examined the dynamics of transcriptional changes of well-established IR-responsive set of genes,including CDKN1A,GADD45A,etc. at 2 and 16 h post-IR,representing early" and "late" radioresponses of hESCs. Our findings suggest the temporal- and hESC line-dependence of stress gene radioresponses with no statistically significant evidence for a linear dose-response relationship within the lowest doses of IR exposures." View Publication