技术资料

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular,human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency,their self-renewal potential,and their ability to create patient-specific cell lines. Unfortunately,pluripotent stem cell-derived CMs are immature,with characteristics more closely resembling fetal CMs than adult CMs,and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation,as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold,compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes,Junctin,CaV1.2,NCX1,HCN4,SERCA2a,Triadin,and CASQ2. Consistent with this,we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine,likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together,these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease. View Publication

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1$$ (HIF-1$$) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1$$ in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study,we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1$$ and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1$$. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats,DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone,leading to bone regeneration. Collectively,the results indicate that DMOG,via activation of the PI3K/Akt pathway,promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration. View Publication

Genome-wide association studies (GWASs) have reported many single nucleotide polymorphisms (SNPs) associated with psychiatric disorders,but knowledge is lacking regarding molecular mechanisms. Here we show that risk alleles spanning multiple genes across the 10q24.32 schizophrenia-related locus are associated in the human brain selectively with an increase in the expression of both BLOC-1 related complex subunit 7 (BORCS7) and a previously uncharacterized,human-specific arsenite methyltransferase (AS3MT) isoform (AS3MT(d2d3)),which lacks arsenite methyltransferase activity and is more abundant in individuals with schizophrenia than in controls. Conditional-expression analysis suggests that BORCS7 and AS3MT(d2d3) signals are largely independent. GWAS risk SNPs across this region are linked with a variable number tandem repeat (VNTR) polymorphism in the first exon of AS3MT that is associated with the expression of AS3MT(d2d3) in samples from both Caucasians and African Americans. The VNTR genotype predicts promoter activity in luciferase assays,as well as DNA methylation within the AS3MT gene. Both AS3MT(d2d3) and BORCS7 are expressed in adult human neurons and astrocytes,and they are upregulated during human stem cell differentiation toward neuronal fates. Our results provide a molecular explanation for the prominent 10q24.32 locus association,including a novel and evolutionarily recent protein that is involved in early brain development and confers risk for psychiatric illness. View Publication

BACKGROUND Alcohol abuse produces an enormous impact on health,society,and the economy. Currently,there are very limited therapies available,largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. RESULTS Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs,but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition,a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover,a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells. CONCLUSIONS This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models. View Publication

Gastric cancer (GC) is one of the most common human cancers. Spheroid colony formation is an effective model for characterization of cancer stem cells. However,gene expression profiles of spheroid colonies obtained from GC cells have not been examined. We performed microarray analyses by Human Genome U133 Plus 2.0 Array in spheroid body-forming and parental cells from MKN-45 and MKN-74 GC cell lines. Kinesin family member C1 (KIFC1) was expressed textgreater2-fold higher in spheroid body-forming cells than in parental cells in both GC lines. Both the number and size of spheres from MKN-45 cells were significantly reduced upon KIFC1 siRNA-transfection compared with negative control siRNA-transfection. Immunohistochemical analysis of 114 GC tissue samples revealed that 42 (37%) of GC cases were positive for KIFC1 expression. GC cases positive for KIFC1 were found more frequently in stage III/IV cases than in stage I/II cases. GC cases positive for KIFC1 were found more frequently in intestinal type GC cases than in diffuse type GC cases. Furthermore,KIFC1-positive GC cases showed high Ki-67 labeling index. Kaplan-Meier analysis demonstrated that KIFC1 expression was not associated with survival. We found positive expression of KIFC1 in CD44‑positive GC and aldehyde dehydrogenase 1 (ALDH1)-positive GC cells. Our results showed that KIFC1 is overexpressed in GC. Since knockdown of KIFC1 inhibited sphere formation,KIFC1 likely plays an important role in cancer stem cells. View Publication

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands,targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features,and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall,the approach described here allows for the rapid generation of human cardiac grafts from hPSCs,in a total of 24 days,providing a suitable platform for cardiac engineering and disease modeling in the human setting. View Publication

Abstract Purpose: The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic,with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells,but previous studies have shown variability in iPSC propensity to differentiate into RPE. Methods: This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid,directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE,which were characterized with respect to global gene expression,expression of RPE markers,and cellular function. Results: We found that all 5 iPSC lines (iPSC-1,iPSC-2,iPSC-3,iPSC-4,and iPSC-12) generated RPE using the directed differentiation protocol; however,2 of the 5 iPSC lines (iPSC-4 and iPSC-... View Publication

On the basis of our previous report verifying that CXCR2 ligands in human placenta-conditioned medium (hPCCM) support human pluripotent stem cell (hPSC) propagation without exogenous bFGF,this study was designed to identify the effect of CXCR2 manipulation on the fate of hPSCs and the underlying mechanism,which had not been previously determined. We observed that CXCR2 inhibition in hPSCs induces predominant differentiation to mesoderm and endoderm with concomitant loss of hPSC characteristics and accompanying decreased expression of mTOR,beta-catenin,and hTERT. These phenomena are recapitulated in hPSCs propagated in conventional culture conditions including bFGF as well as those in hPCCM without exogenous bFGF,suggesting that the action of CXCR2 on hPSCs might not be associated with a bFGF-related mechanism. In addition,the specific CXCR2 ligand GROalpha markedly increased the expression of ectodermal markers in differentiation-committed embryoid bodies derived from hPSCs. This finding suggests that CXCR2 inhibition in hPSCs prohibits the propagation of hPSCs and leads to predominant differentiation to mesoderm and endoderm owing to the blockage of ectodermal differentiation. Taken together,our results indicate that CXCR2 preferentially supports the maintenance of hPSC characteristics as well as facilitates ectodermal differentiation after the commitment to differentiation,and that the mechanism might be associated with mTOR,beta-catenin,and hTERT activities. View Publication

BACKGOUND The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson's disease (PD). METHODS The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD,using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study,patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson's Disease Rating Scale (UPDRS) to measure clinical symptoms. RESULTS The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC,consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; ptextless0.05 for all values) in the PD group treated with NAC,and no measurable changes in the control group. UPDRS scores were also significantly improved in the NAC group (mean improvement of 12.9%,p = 0.01). CONCLUSIONS The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients,and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. TRIAL REGISTRATION ClinicalTrials.gov NCT02445651. View Publication

Human iPSC line L749.1 was generated from fibroblasts of a patient with Leigh syndrome associated with a heteroplasmic mutation in the MT-ATP6 gene. Reprogramming factors OCT4,SOX2,CMYC and KLF4 were delivered using retroviruses. View Publication

Human embryonic stem cell line BJNhem20-pCAG-tdTomato was generated using non-viral method. The construct pCAG-tdTomato was transfected using microporation procedure. This fluorescent hESC line can help to study heterogeneity within individual cells in hESC colonies by enabling live tracking of their growth,migration and differentiation properties. This cell line also serves as a resource for additional transgene introduction/knock-out/knock-in generation in a fluorescent background and allows ease of analysis in studies involving cell mixing. View Publication

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30,reported to be a biomarker of hESCs with abnormal karyotype,in undifferentiated and spontaneously differentiated hESC.backslashnbackslashnMETHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC,even at very early passages,without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC.backslashnbackslashnCONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions,and that KO-SR may play a role. In addition,the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities. View Publication