技术资料

Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt,correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart,we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb,with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency,consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites,we observed a high degree of gene excisions and inversions,which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency,deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments,particularly in human iPSC. View Publication

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here,we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable,and endothelial progenitor differentiation was β-catenin dependent. Furthermore,by clonal analysis,we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent,capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings,formed tube-like structures,imported acetylated low-density lipoprotein,and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs. View Publication

Human pluripotent stem cells (hPSCs) - including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) - are very promising candidates for cell therapies,tissue engineering,high throughput pharmacology screens,and toxicity testing. These applications require large numbers of high quality cells; however,scalable production of human pluripotent stem cells and their derivatives at a high density and under well-defined conditions has been a challenge. We recently reported a simple,efficient,fully defined,scalable,and good manufacturing practice (GMP) compatible 3D culture system based on a thermoreversible hydrogel for hPSC expansion and differentiation. Here,we describe additional design rationale and characterization of this system. For instance,we have determined that culturing hPSCs as a suspension in a liquid medium can exhibit lower volumetric yields due to cell agglomeration and possible shear force-induced cell loss. By contrast,using hydrogels as 3D scaffolds for culturing hPSCs reduces aggregation and may insulate from shear forces. Additionally,hydrogel-based 3D culture systems can support efficient hPSC expansion and differentiation at a high density if compatible with hPSC biology. Finally,there are considerable opportunities for future development to further enhance hydrogel-based 3D culture systems for producing hPSCs and their progeny. View Publication

Tendon-bone junctions (TBJs) are frequently injured,especially in athletic settings. Healing of TBJ injuries is slow and is often repaired with scar tissue formation that compromises normal function. This study explored the feasibility of using kartogenin (KGN),a biocompound,to enhance the healing of injured TBJs. We first determined the effects of KGN on the proliferation and chondrogenic differentiation of rabbit bone marrow stromal cells (BMSCs) and patellar tendon stem/progenitor cells (PTSCs) in vitro. KGN enhanced cell proliferation in both cell types in a concentration-dependent manner and induced chondrogenic differentiation of stem cells,as demonstrated by high expression levels of chondrogenic markers aggrecan,collagen II and Sox-9. Besides,KGN induced the formation of cartilage-like tissues in cell cultures,as observed through the staining of abundant proteoglycans,collagen II and osteocalcin. When injected into intact rat patellar tendons in vivo,KGN induced cartilage-like tissue formation in the injected area. Similarly,when KGN was injected into experimentally injured rat Achilles TBJs,wound healing in the TBJs was enhanced,as evidenced by the formation of extensive cartilage-like tissues. These results suggest that KGN may be used as an effective cell-free clinical therapy to enhance the healing of injured TBJs. View Publication

We have identified a rare coding mutation,T835M (rs137875858),in the UNC5C netrin receptor gene that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer's disease and that was associated with disease across four large case-control cohorts (odds ratio = 2.15,Pmeta = 0.0095). T835M alters a conserved residue in the hinge region of UNC5C,and in vitro studies demonstrate that this mutation leads to increased cell death in human HEK293T cells and in rodent neurons. Furthermore,neurons expressing T835M UNC5C are more susceptible to cell death from multiple neurotoxic stimuli,including $\beta$-amyloid (A$\beta$),glutamate and staurosporine. On the basis of these data and the enriched hippocampal expression of UNC5C in the adult nervous system,we propose that one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer's disease is by increasing susceptibility to neuronal cell death,particularly in vulnerable regions of the Alzheimer's disease brain. View Publication

The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here,neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1,Sox2,Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains,respectively,of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes,epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together,these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays. View Publication

BACKGROUND Induced pluripotent stem cells (iPSCs) hold tremendous potential,both as a biological tool to uncover the pathophysiology of disease by creating relevant human cell models and as a source of cells for cell-based therapeutic applications. Studying the reprogramming process will also provide significant insight into tissue development. OBJECTIVE We sought to characterize the derivation of iPSC lines from nasal epithelial cells (NECs) isolated from nasal mucosa samples of children,a highly relevant and easily accessible tissue for pediatric populations. METHODS We performed detailed comparative analysis on the transcriptomes and methylomes of NECs,iPSCs derived from NECs (NEC-iPSCs),and embryonic stem cells (ESCs). RESULTS NEC-iPSCs express pluripotent cell markers,can differentiate into all 3 germ layers in vivo and in vitro,and have a transcriptome and methylome remarkably similar to those of ESCs. However,residual DNA methylation marks exist,which are differentially methylated between NEC-iPSCs and ESCs. A subset of these methylation markers related to epithelium development and asthma and specific to NEC-iPSCs persisted after several passages in vitro,suggesting the retention of an epigenetic memory of their tissue of origin. Our analysis also identified novel candidate genes with dynamic gene expression and DNA methylation changes during reprogramming,which are indicative of possible roles in airway epithelium development. CONCLUSION NECs are an excellent tissue source to generate iPSCs in pediatric asthmatic patients,and detailed characterization of the resulting iPSC lines would help us better understand the reprogramming process and retention of epigenetic memory. View Publication

Early cognitive deficits in Alzheimer's disease (AD) seem to be correlated to dysregulation of glutamate receptors evoked by amyloid-beta (Aβ) peptide. Aβ interference with the activity of N-methyl-d-aspartate receptors (NMDARs) may be a relevant factor for Aβ-induced mitochondrial toxicity and neuronal dysfunction. To evaluate the role of mitochondria in NMDARs activation mediated by Aβ,we followed in situ single-cell simultaneous measurement of cytosolic free Ca(2+)(Cai(2+)) and mitochondrial membrane potential in primary cortical neurons. Our results show that direct exposure to Aβ + NMDA largely increased Cai(2+) and induced immediate mitochondrial depolarization,compared with Aβ or NMDA alone. Mitochondrial depolarization induced by rotenone strongly inhibited the rise in Cai(2+) evoked by Aβ or NMDA,suggesting that mitochondria control Ca(2+) entry through NMDARs. However,incubation with rotenone did not preclude mitochondrial Ca(2+) (mitCa(2+)) retention in cells treated with Aβ. Aβ-induced Cai(2+) and mitCa(2+) rise were inhibited by ifenprodil,an antagonist of GluN2B-containing NMDARs. Exposure to Aβ + NMDA further evoked a higher mitCa(2+) retention,which was ameliorated in GluN2B(-/-) cortical neurons,largely implicating the involvement of this NMDAR subunit. Moreover,pharmacologic inhibition of endoplasmic reticulum (ER) inositol-1,4,5-triphosphate receptor (IP3R) and mitCa(2+) uniporter (MCU) evidenced that Aβ + NMDA-induced mitCa(2+) rise involves ER Ca(2+) release through IP3R and mitochondrial entry by the MCU. Altogether,data highlight mitCa(2+) dyshomeostasis and subsequent dysfunction as mechanisms relevant for early neuronal dysfunction in AD linked to Aβ-mediated GluN2B-composed NMDARs activation. View Publication

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis,and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein,we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly,TRAIL treatment induced RANKL mRNA expression in these cells. In addition,TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition,siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus,our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells. View Publication

The neplanocin A analogue 3-deazaneplanocin A (2b) has been synthesized. A direct SN2 displacement on the cyclopentenyl mesylate 3 by the sodium salt of 6-chloro-3-deazapurine afforded the desired regioisomer 4 as the major product. After deprotection,this material was converted to 3-deazaneplanocin A in two steps. X-ray crystallographic analysis confirmed the assigned structure. Consistent with its potent inhibition of S-adenosylhomocysteine hydrolase,3-deazaneplanocin A displayed excellent antiviral activity in cell culture against vesicular stomatitis,parainfluenza type 3,yellow fever,and vaccinia viruses. Antiviral activity was also displayed in vivo against vaccinia virus by using a mouse tailpox assay. The significantly lower cytotoxicity of 3-deazaneplanocin A,relative to its parent compound neplanocin A,may be due to its lack of conversion to 5'-triphosphate and S-adenosylmethionine metabolites. View Publication

OBJECTIVE Charcot-Marie-Tooth (CMT) disease is a group of inherited peripheral neuropathies associated with mutations or copy number variations in over 70 genes encoding proteins with fundamental roles in the development and function of Schwann cells and peripheral axons. Here,we used iPSC-derived cells to identify common pathophysiological mechanisms in axonal CMT. METHODS iPSC lines from patients with two distinct forms of axonal CMT (CMT2A and CMT2E) were differentiated into spinal cord motor neurons and used to study axonal structure and function and electrophysiological properties in vitro. RESULTS iPSC-derived motor neurons exhibited gene and protein expression,ultrastructural and electrophysiological features of mature primary spinal cord motor neurons. Cytoskeletal abnormalities were found in neurons from a CMT2E (NEFL) patient and corroborated by a mouse model of the same NEFL point mutation. Abnormalities in mitochondrial trafficking were found in neurons derived from this patient,but were only mildly present in neurons from a CMT2A (MFN2) patient. Novel electrophysiological abnormalities,including reduced action potential threshold and abnormal channel current properties were observed in motor neurons derived from both of these patients. INTERPRETATION Human iPSC-derived motor neurons from axonal CMT patients replicated key pathophysiological features observed in other models of MFN2 and NEFL mutations,including abnormal cytoskeletal and mitochondrial dynamics. Electrophysiological abnormalities found in axonal CMT iPSC-derived human motor neurons suggest that these cells are hyperexcitable and have altered sodium and calcium channel kinetics. These findings may provide a new therapeutic target for this group of heterogeneous inherited neuropathies. View Publication

The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However,the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here,we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC),an extracellular matrix protein associated with HSC niches,strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders,including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential. View Publication