技术资料

The future of safe cell-based therapy rests on overcoming teratoma/tumor formation,in particular when using human pluripotent stem cells (hPSCs),such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation,complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile,we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e.,survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors,leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular,a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g.,quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast,differentiated cell types (e.g.,dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together,these results provide the proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy." View Publication

INTRODUCTION: Ischemic stroke is a leading cause of death and disability,but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study,we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model.backslashnbackslashnMETHODS: Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention.backslashnbackslashnRESULTS: After 11 days of neural induction by using the small-molecule protocol,over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude,repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals.backslashnbackslashnCONCLUSIONS: Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo,enhance regenerative activities,and improve sensory recovery after ischemic stroke. View Publication

Sickle cell disease (SCD) is the most common human genetic disease which is caused by a single mutation of human β-globin (HBB) gene. The lack of long-term treatment makes the development of reliable cell and gene therapies highly desirable. Disease-specific patient-derived human induced pluripotent stem cells (hiPSCs) have great potential for developing novel cell and gene therapies. With the disease-causing mutations corrected in situ,patient-derived hiPSCs can restore normal cell functions and serve as a renewable autologous cell source for the treatment of genetic disorders. Here we successfully utilized transcription activator-like effector nucleases (TALENs),a recently emerged novel genome editing tool,to correct the SCD mutation in patient-derived hiPSCs. The TALENs we have engineered are highly specific and generate minimal off-target effects. In combination with piggyBac transposon,TALEN-mediated gene targeting leaves no residual ectopic sequences at the site of correction and the corrected hiPSCs retain full pluripotency and a normal karyotype. Our study demonstrates an important first step of using TALENs for the treatment of genetic diseases such as SCD,which represents a significant advance toward hiPSC-based cell and gene therapies. View Publication

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence,which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here,we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity,simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. textcopyright 2013 Elsevier Inc. View Publication

Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2,ApoE3,and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD),ApoE3 is neutral,and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis,however,remains unclear. Using ES-cell-derived human neurons,we show that ApoE secreted by glia stimulates neuronal Aβ production with an ApoE4< ApoE3< ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK),a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation,stimulating the transcription factor AP-1,which in turn enhances transcription of amyloid-β precursor protein (APP) and thereby increases amyloid-β levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis. View Publication

Although gene-gene interaction,or epistasis,plays a large role in complex traits in model organisms,genome-wide by genome-wide searches for two-way interaction have limited power in human studies. We thus used knowledge of a biological pathway in order to identify a contribution of epistasis to autism spectrum disorders (ASDs) in humans,a reverse-pathway genetic approach. Based on previous observation of increased ASD symptoms in Mendelian disorders of the Ras/MAPK pathway (RASopathies),we showed that common SNPs in RASopathy genes show enrichment for association signal in GWAS (P = 0.02). We then screened genome-wide for interactors with RASopathy gene SNPs and showed strong enrichment in ASD-affected individuals (P < 2.2 x 10-16),with a number of pairwise interactions meeting genome-wide criteria for significance. Finally,we utilized quantitative measures of ASD symptoms in RASopathy-affected individuals to perform modifier mapping via GWAS. One top region overlapped between these independent approaches,and we showed dysregulation of a gene in this region,GPR141,in a RASopathy neural cell line. We thus used orthogonal approaches to provide strong evidence for a contribution of epistasis to ASDs,confirm a role for the Ras/MAPK pathway in idiopathic ASDs,and to identify a convergent candidate gene that may interact with the Ras/MAPK pathway. View Publication

BACKGROUND Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is,containing both endothelial cells (ECs) and cardiomyocytes. METHODS We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP),and an Akt-activated EC line (E4(+)ECs). We quantified spontaneous beating rates,synchrony,and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. RESULTS After 8 days in culture,94% ± 6% of the NKX2-5GFP(+) cells were beating when hESCs embryonic bodies were plated on E4(+)ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP(+) cardiomyocytes were close to the E4(+)ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network,as illustrated by the loss of synchronization upon the disruption of endothelial bridges. CONCLUSIONS The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication