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BACKGROUND Congenital human cytomegalovirus (HCMV) infection,a leading cause of birth defects,is most often manifested as neurological disorders. The pathogenesis of HCMV-induced neurological disorders is,however,largely unresolved,primarily because of limited availability of model systems to analyze the effects of HCMV infection on neural cells. METHODS An induced pluripotent stem cell (iPSC) line was established from the human fibroblast line MRC5 by introducing the Yamanaka's four factors and then induced to differentiate into neural stem/progenitor cells (NSPCs) by dual inhibition of the SMAD signaling pathway using Noggin and SB-431542. RESULTS iPSC-derived NSPCs (NSPC/iPSCs) were susceptible to HCMV infection and allowed the expression of both early and late viral gene products. HCMV-infected NSPC/iPSCs underwent apoptosis with the activation of caspase-3 and -9 as well as positive staining by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Cytochrome c release from mitochondria to cytosol was observed in these cells,indicating the involvement of mitochondrial dysfunction in their apoptosis. In addition,phosphorylation of proteins involved in the unfolded protein response (UPR),such as PKR-like eukaryotic initiation factor 2a kinase (PERK),c-Jun NH2-terminal kinase (JNK),inositol-requiring enzyme 1 (IRE1),and the alpha subunit of eukaryotic initiation factor 2 (eIF2$$) was observed in HCMV-infected NSPC/iPSCs. These results,coupled with the finding of increased expression of mRNA encoding the C/EBP-homologous protein (CHOP) and the detection of a spliced form of X-box binding protein 1 (XBP1) mRNA,suggest that endoplasmic reticulum (ER) stress is also involved in HCMV-induced apoptosis of these cells. CONCLUSIONS iPSC-derived NSPCs are thought to be a useful model to study HCMV neuropathogenesis and to analyze the mechanisms of HCMV-induced apoptosis in neural cells. View Publication

Transactive response DNA-binding protein 43 (TDP-43) is a major pathological protein in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). There are many disease-associated mutations in TDP-43,and several cellular and animal models with ectopic overexpression of mutant TDP-43 have been established. Here we sought to study altered molecular events in FTD and ALS by using induced pluripotent stem cell (iPSC) derived patient neurons. We generated multiple iPSC lines from an FTD/ALS patient with the TARDBP A90V mutation and from an unaffected family member who lacked the mutation. After extensive characterization,two to three iPSC lines from each subject were selected,differentiated into postmitotic neurons,and screened for relevant cell-autonomous phenotypes. Patient-derived neurons were more sensitive than control neurons to 100 nM straurosporine but not to other inducers of cellular stress. Three disease-relevant cellular phenotypes were revealed under staurosporine-induced stress. First,TDP-43 was localized in the cytoplasm of a higher percentage of patient neurons than control neurons. Second,the total TDP-43 level was lower in patient neurons with the A90V mutation. Third,the levels of microRNA-9 (miR-9) and its precursor pri-miR-9-2 decreased in patient neurons but not in control neurons. The latter is likely because of reduced TDP-43,as shRNA-mediated TDP-43 knockdown in rodent primary neurons also decreased the pri-miR-9-2 level. The reduction in miR-9 expression was confirmed in human neurons derived from iPSC lines containing the more pathogenic TARDBP M337V mutation,suggesting miR-9 downregulation might be a common pathogenic event in FTD/ALS. These results show that iPSC models of FTD/ALS are useful for revealing stress-dependent cellular defects of human patient neurons containing rare TDP-43 mutations in their native genetic contexts. View Publication

Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident with human pluripotent stem cells (hPSCs): microenvironmental heterogeneity limits hPSC cell fate control. We developed a high-throughput platform to screen hPSCs in configurable microenvironments in which we optimized colony size,cell density and other parameters to achieve rapid and robust cell fate responses to exogenous cues. We used this platform to perform single-cell protein expression profiling,revealing that Oct4 and Sox2 costaining discriminates pluripotent,neuroectoderm,primitive streak and extraembryonic cell fates. We applied this Oct4-Sox2 code to analyze dose responses of 27 developmental factors to obtain lineage-specific concentration optima and to quantify cell line–specific endogenous signaling pathway activation and differentiation bias. We demonstrated that short-term responses predict definitive endoderm induction efficiency and can be used to rescue differentiation of cell lines reticent to cardiac induction. This platform will facilitate high-throughput hPSC-based screening and quantification of lineage-induction bias. View Publication

Biochemical factors can help reprogram somatic cells into pluripotent stem cells,yet the role of biophysical factors during reprogramming is unknown. Here,we show that biophysical cues,in the form of parallel microgrooves on the surface of cell-adhesive substrates,can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically,decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves,suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications. View Publication

Geometric factors including the size,shape,density,and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such,there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here,we demonstrate a simple,robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size,density,and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages,geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person,an increase in differentiation yield,a faster experimental workflow,and a simpler protocol to communicate and follow. Furthermore,the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening. 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

A metabolic biomarker-based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery-phase detection of potential human developmental toxicants. In this study,metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure-based biomarker assay using these metabolites,along with a cytotoxicity endpoint,was then developed using a 9-point dose–response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity,an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy,but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity,100% specificity). The assay had a high concordance (≥75%) with existing in vivo models,demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests. View Publication

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies,disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro 'patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT),an inherited form of fatal arrhythmia. Here,we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs,both in resting conditions and after $\$-adrenergic stimulation,resembling the cardiac phenotype of the patients. Furthermore,treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine),an antiarrhythmic drug that inhibits Ca(2+)/calmodulin-dependent serine-threonine protein kinase II (CaMKII),drastically reduced the presence of DADs in CVPT-CMs,rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition,intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients,whereas in the wild-type clusters,only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice,the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells,supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies. 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

Maternal-effect mutations in NLRP7 cause rare biparentally inherited hydatidiform moles (BiHMs),abnormal pregnancies containing hypertrophic vesicular trophoblast but no embryo. BiHM trophoblasts display abnormal DNA methylation patterns affecting maternally methylated germline differentially methylated regions (gDMRs),suggesting that NLRP7 plays an important role in reprogramming imprinted gDMRs. How NLRP7—a component of the CATERPILLAR family of proteins involved in innate immunity and apoptosis—causes these specific DNA methylation and trophoblast defects is unknown. Because rodents lack NLRP7,we used human embryonic stem cells to study its function and demonstrate that NLRP7 interacts with YY1,an important chromatin-binding factor. Reduced NLRP7 levels alter DNA methylation and accelerate trophoblast lineage differentiation. NLRP7 thus appears to function in chromatin reprogramming and DNA methylation in the germline or early embryonic development,functions not previously associated with members of the NLRP family. View Publication

Background Human embryonic stem cells (hESCs) serve as a potential unlimited ex vivo source of cardiomyocytes for disease modeling,cardiotoxicity screening,drug discovery,and cell-based therapies. Despite the fundamental importance of Ca2+-induced Ca2+ release in excitation-contraction coupling,the mechanistic basis of Ca2+ handling of hESC-derived ventricular cardiomyocytes (VCMs) remains elusive. Objectives To study Ca2+ sparks as unitary events of Ca2+ handling for mechanistic insights. Methods To avoid ambiguities owing to the heterogeneous nature,we experimented with hESC-VCMs,purified on the basis of zeocin resistance and signature ventricular action potential after LV-MLC2v-tdTomato-T2A-Zeo transduction. Results Ca2+ sparks that were sensitive to inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (thapsigargin and cyclopiazonic acid) and ryanodine receptor (RyR; ryanodine,tetracaine) but not inositol trisphosphate receptors (xestospongin C and 2-aminoethyl diphenylborinate) could be recorded. In a permeabilization model,we further showed that RyRs could be sensitized by Ca2+. Increasing external Ca2+ dramatically escalated the basal Ca2+ and spark frequency. Furthermore,RyR-mediated Ca2+ release sensitized nearby RyRs,leading to compound Ca2+ sparks. Depolarization or L-type Ca2+ channel agonist (FPL 64176 and Bay K8644) pretreatment induced an extracellular Ca2+-dependent cytosolic Ca2+ increase and reduced the sarcoplasmic reticulum content. By contrast,removal of external Na+ or the addition of the Na+-Ca2+ exchanger inhibitor (KB-R7943 and SN-6) had no effect,suggesting that the Na+-Ca2+ exchanger is not involved in triggering sparks. Inhibition of mitochondrial Ca2+ uptake by carbonyl cyanide m-chlorophenyl hydrazone promoted Ca2+ waves. Conclusion Taken collectively,our findings provide the first lines of direct evidence that hESC-VCMs have functional Ca2+-induced Ca2+ release. However,the sarcoplasmic reticulum is leaky and without a mature terminating mechanism in early development. 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