技术资料

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here,we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes,including tal1/scl,gata1,gata2,hoxB4,and lhx2,into CM ESCs. By immunochemical and morphological analyses,we demonstrated that overexpression of tal1/scl,but not the remaining genes,dramatically increased hematopoiesis of CM ESCs,resulting in multiple blood-cell lineages. Furthermore,flow cytometric analysis demonstrated that CD34,a hematopoietic stem/progenitor cell marker,was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells. View Publication

Definitive mesoderm arises from a bipotent mesendodermal population,and to study processes controlling its development at this stage,embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context,we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively,EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation,EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic,vascular,and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition,the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context. View Publication

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings,however,requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study,we show that once specified to neuro-epithelial lineage,hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However,our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes,namely,migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes,a new potential source for therapeutic use for brain tumors and other neurological diseases. 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

The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis,we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro,we generated neurons harboring disease-causing mutations from patient-specific,induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons,reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro,including oxidative stress vulnerability and altered neuronal activity. Notably,PD neurons show that elevated RBFOX1,a gene previously linked to neurodevelopmental diseases,underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes. View Publication

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body,including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system,provides a platform for the screening of chemical libraries that affect these processes,and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However,defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner,with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells,exhibit spontaneous electrical activity,and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months,even without astrocyte feeder layers. 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

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical,environmental,and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part,previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation,which are highly nonphysiologic,or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair,we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs,compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus,the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny. 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

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

Objective Friedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat expansion disorder caused by epigenetic silencing of the frataxin gene (FXN). Current research suggests that damage and variation of mitochondrial DNA (mtDNA) contribute to the molecular pathogenesis of FRDA. We sought to establish the extent of the mutation burden across the mitochondrial genome in FRDA cells and investigate the molecular mechanisms connecting FXN downregulation and the acquisition of mtDNA damage. Methods Damage and mutation load in mtDNA of a panel of FRDA and control fibroblasts were determined using qPCR and next-generation MiSeq sequencing,respectively. The capacity of FRDA and control cells to repair oxidative lesions in their mtDNA was measured using a quantitative DNA damage assay. Comprehensive RNA sequencing gene expression analyses were conducted to assess the status of DNA repair and metabolism genes in FRDA cells. Results Acute or prolonged downregulation of FXN expression resulted in a significant increase in mtDNA damage that translated to a significant elevation of mutation load in mtDNA. The predominant mutations identified throughout the mtDNA were CtextgreaterT,GtextgreaterA transitions (P = 0.007). Low FXN expression reduced capacity to repair oxidative damage in mtDNA. Downregulation of FXN expression strongly correlated (r = 0.73) with decreased levels of base excision repair (BER) DNA glycosylase NTHL1. Interpretation Downregulation of FXN expression in FRDA cells elevates mtDNA damage,increases mutation load of the mitochondrial genome,and diminishes DNA repair capacity. Progressive accumulation of mtDNA mutations in vulnerable FRDA patient cells reduces mitochondrial fitness ultimately leading to cell death. 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