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Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 64-year old male Parkinson's disease (PD) patient with N551K variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model can complement in vivo PD models for pathophysiological studies and drug screening. View Publication

Human iPSC line Oex2054SV.4 was generated from fibroblasts of a patient with an optic atrophy 'plus' phenotype associated with a heterozygous mutation in the OPA1 gene. Reprogramming factors OCT3/4,SOX2,CMYC and KLF4 were delivered using a non-integrative methodology that involves the use of Sendai virus. View Publication

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous,de novo,premature termination codon (PTC) mutation (NM024757.4(EHMT1):c.3413GtextgreaterA; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore,the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome,also for drug testing,early biomarker discovery and gene therapy studies. View Publication

There is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust,patient-specific tissue model systems for studying the pathogenesis of vascular disease,and for developing novel therapeutic interventions. Here,we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore,we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system,extendable to study other vascular proliferative diseases for drug screening. Thus,this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications. View Publication

Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare,early-onset retinal dystrophy characterised by distinct bands of circumferential pigmentary degeneration in the peripheral retina and developmental eye defects. ADVIRC is caused by mutations in the Bestrophin1 (BEST1) gene,which encodes a transmembrane protein thought to function as an ion channel in the basolateral membrane of retinal pigment epithelial (RPE) cells. Previous studies suggest that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. Here,we have used induced pluripotent stem cell (iPSC) technology to investigate the effects of an ADVIRC associated BEST1 mutation (c.704T textgreater C,p.V235A) in patient-derived iPSC-RPE. We found no evidence of alternate splicing of the BEST1 transcript in ADVIRC iPSC-RPE,however in patient-derived iPSC-RPE,BEST1 was expressed at the basolateral membrane and the apical membrane. During human eye development we show that BEST1 is expressed more abundantly in peripheral RPE compared to central RPE and is also expressed in cells of the developing retina. These results suggest that higher levels of mislocalised BEST1 expression in the periphery,from an early developmental stage,could provide a mechanism that leads to the distinct clinical phenotype observed in ADVIRC patients. View Publication

BACKGROUND Individuals with 22q11.2 Deletion Syndrome (22q11.2 DS) are a specific high-risk group for developing schizophrenia (SZ),schizoaffective disorder (SAD) and autism spectrum disorders (ASD). Several genes in the deleted region have been implicated in the development of SZ,e.g.,PRODH and DGCR8. However,the mechanistic connection between these genes and the neuropsychiatric phenotype remains unclear. To elucidate the molecular consequences of 22q11.2 deletion in early neural development,we carried out RNA-seq analysis to investigate gene expression in early differentiating human neurons derived from induced pluripotent stem cells (iPSCs) of 22q11.2 DS SZ and SAD patients. METHODS Eight cases (ten iPSC-neuron samples in total including duplicate clones) and seven controls (nine in total including duplicate clones) were subjected to RNA sequencing. Using a systems level analysis,differentially expressed genes/gene-modules and pathway of interests were identified. Lastly,we related our findings from in vitro neuronal cultures to brain development by mapping differentially expressed genes to BrainSpan transcriptomes. RESULTS We observed ˜2-fold reduction in expression of almost all genes in the 22q11.2 region in SZ (37 genes reached p-value textless 0.05,36 of which reached a false discovery rate textless 0.05). Outside of the deleted region,745 genes showed significant differences in expression between SZ and control neurons (p textless 0.05). Function enrichment and network analysis of the differentially expressed genes uncovered converging evidence on abnormal expression in key functional pathways,such as apoptosis,cell cycle and survival,and MAPK signaling in the SZ and SAD samples. By leveraging transcriptome profiles of normal human brain tissues across human development into adulthood,we showed that the differentially expressed genes converge on a sub-network mediated by CDC45 and the cell cycle,which would be disrupted by the 22q11.2 deletion during embryonic brain development,and another sub-network modulated by PRODH,which could contribute to disruption of brain function during adolescence. CONCLUSIONS This study has provided evidence for disruption of potential molecular events in SZ patient with 22q11.2 deletion and related our findings from in vitro neuronal cultures to functional perturbations that can occur during brain development in SZ. View Publication

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene,resulting in absent or defective gp91(phox) protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation,we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients,which restored gp91(phox) expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed,involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91(phox) expression or ROS activity in iPSC-derived granulocytes. In contrast,targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91(phox) and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore,it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression. View Publication

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently,there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS,and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study,we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity,low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS,treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span,in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms,including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases. View Publication

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here,we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4,SOX2,KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology,chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65,RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane,and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together,our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases. View Publication

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSCs) hold great potential for the modelling of these diseases or as potential therapeutic agents. However,to fulfill this promise,a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina,in particular the presence of photoreceptors with outer-segment discs and light sensitivity. Here we report that hiPSC can,in a highly autonomous manner,recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form three-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover,the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation,showing the beginning of outer-segment disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modelling and open possibilities for future therapies. View Publication

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function,little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet,understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore,we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs,from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties,including resting membrane potential,action potential,sodium and potassium channel currents,somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons,the resting membrane potential became more negative,the expression of voltage-gated sodium channels increased,the membrane became capable of generating action potentials following adequate depolarization and,at day 48-55,50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step,of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology,as electrophysiological properties of iPSC-derived neurons mature over time. View Publication

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature,differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF,NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases. View Publication