技术资料

Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials,we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg,cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence,their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose,the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4-40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages,the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4,SSEA3,and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM,however,hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications,providing their degradation rate is moderate. Additionally,the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro,and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds. View Publication

BACKGROUND Congenital human cytomegalovirus (HCMV) infection,a leading cause of birth defects,is most often manifested as neurological disorders. The pathogenesis of HCMV-induced neurological disorders is,however,largely unresolved,primarily because of limited availability of model systems to analyze the effects of HCMV infection on neural cells. METHODS An induced pluripotent stem cell (iPSC) line was established from the human fibroblast line MRC5 by introducing the Yamanaka's four factors and then induced to differentiate into neural stem/progenitor cells (NSPCs) by dual inhibition of the SMAD signaling pathway using Noggin and SB-431542. RESULTS iPSC-derived NSPCs (NSPC/iPSCs) were susceptible to HCMV infection and allowed the expression of both early and late viral gene products. HCMV-infected NSPC/iPSCs underwent apoptosis with the activation of caspase-3 and -9 as well as positive staining by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Cytochrome c release from mitochondria to cytosol was observed in these cells,indicating the involvement of mitochondrial dysfunction in their apoptosis. In addition,phosphorylation of proteins involved in the unfolded protein response (UPR),such as PKR-like eukaryotic initiation factor 2a kinase (PERK),c-Jun NH2-terminal kinase (JNK),inositol-requiring enzyme 1 (IRE1),and the alpha subunit of eukaryotic initiation factor 2 (eIF2$$) was observed in HCMV-infected NSPC/iPSCs. These results,coupled with the finding of increased expression of mRNA encoding the C/EBP-homologous protein (CHOP) and the detection of a spliced form of X-box binding protein 1 (XBP1) mRNA,suggest that endoplasmic reticulum (ER) stress is also involved in HCMV-induced apoptosis of these cells. CONCLUSIONS iPSC-derived NSPCs are thought to be a useful model to study HCMV neuropathogenesis and to analyze the mechanisms of HCMV-induced apoptosis in neural cells. View Publication

Transactive response DNA-binding protein 43 (TDP-43) is a major pathological protein in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). There are many disease-associated mutations in TDP-43,and several cellular and animal models with ectopic overexpression of mutant TDP-43 have been established. Here we sought to study altered molecular events in FTD and ALS by using induced pluripotent stem cell (iPSC) derived patient neurons. We generated multiple iPSC lines from an FTD/ALS patient with the TARDBP A90V mutation and from an unaffected family member who lacked the mutation. After extensive characterization,two to three iPSC lines from each subject were selected,differentiated into postmitotic neurons,and screened for relevant cell-autonomous phenotypes. Patient-derived neurons were more sensitive than control neurons to 100 nM straurosporine but not to other inducers of cellular stress. Three disease-relevant cellular phenotypes were revealed under staurosporine-induced stress. First,TDP-43 was localized in the cytoplasm of a higher percentage of patient neurons than control neurons. Second,the total TDP-43 level was lower in patient neurons with the A90V mutation. Third,the levels of microRNA-9 (miR-9) and its precursor pri-miR-9-2 decreased in patient neurons but not in control neurons. The latter is likely because of reduced TDP-43,as shRNA-mediated TDP-43 knockdown in rodent primary neurons also decreased the pri-miR-9-2 level. The reduction in miR-9 expression was confirmed in human neurons derived from iPSC lines containing the more pathogenic TARDBP M337V mutation,suggesting miR-9 downregulation might be a common pathogenic event in FTD/ALS. These results show that iPSC models of FTD/ALS are useful for revealing stress-dependent cellular defects of human patient neurons containing rare TDP-43 mutations in their native genetic contexts. View Publication

Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident with human pluripotent stem cells (hPSCs): microenvironmental heterogeneity limits hPSC cell fate control. We developed a high-throughput platform to screen hPSCs in configurable microenvironments in which we optimized colony size,cell density and other parameters to achieve rapid and robust cell fate responses to exogenous cues. We used this platform to perform single-cell protein expression profiling,revealing that Oct4 and Sox2 costaining discriminates pluripotent,neuroectoderm,primitive streak and extraembryonic cell fates. We applied this Oct4-Sox2 code to analyze dose responses of 27 developmental factors to obtain lineage-specific concentration optima and to quantify cell line–specific endogenous signaling pathway activation and differentiation bias. We demonstrated that short-term responses predict definitive endoderm induction efficiency and can be used to rescue differentiation of cell lines reticent to cardiac induction. This platform will facilitate high-throughput hPSC-based screening and quantification of lineage-induction bias. View Publication

A metabolic biomarker-based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery-phase detection of potential human developmental toxicants. In this study,metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure-based biomarker assay using these metabolites,along with a cytotoxicity endpoint,was then developed using a 9-point dose–response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity,an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy,but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity,100% specificity). The assay had a high concordance (≥75%) with existing in vivo models,demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests. View Publication

Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However,the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here,we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition,manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally,analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively,our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs. ?? 2013 The Authors. View Publication

BACKGROUND: Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired,resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However,the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore,a human model using patient-specific induced pluripotent stem cells provides a promising approach. METHODS: We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4,Klf4,Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation. RESULTS: The hiPSCs expressed different stem cell markers,e.g. Nanog,Tra-1-81 and SSEA4. Using the embryoid body assay,the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice,demonstrating their pluripotency. In addition,neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels,spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient,derived hiPSCs,and neural progenitor cells,but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay,demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient. CONCLUSIONS: We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts,providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease. View Publication

Diblock copolymers consisting of poly(ethylene glycol)-block-poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-l-glutamate) (PEG-b-PVBLG-8) were synthesized and evaluated for their ability to mediate gene delivery in hard-to-transfect cells like IMR-90 human fetal lung fibroblasts and human embryonic s View Publication

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells,capable of germline transmission,from mouse somatic cells by transduction of four defined transcription factors. Here,we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4,and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology,proliferation,surface antigens,gene expression,epigenetic status of pluripotent cell-specific genes,and telomerase activity. Furthermore,these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts. View Publication

Recently,a number of clinical trials used either mesenchymal stem cells (MSCs) or natural killer (NK) cells in an attempt to improve the effectiveness of hematopoietic stem cell transplantation (HSCT). In view of the relevant role of both MSCs and NK cells in HSCT,we have recently explored the result of possible interactions between the 2 cell types. We found that activated NK cells could kill MSCs,whereas MSCs strongly inhibited interleukin-2 (IL-2)-induced NK-cell proliferation. In this study,we further analyzed the inhibitory effect exerted by MSCs on NK cells. We show that MSCs not only inhibit the cytokine-induced proliferation of freshly isolated NK cells but also prevent the induction of effector functions,such as cytotoxic activity and cytokine production. Moreover,we show that this inhibitory effect is related to a sharp down-regulation of the surface expression of the activating NK receptors NKp30,NKp44,and NKG2D. Finally,we demonstrate that indoleamine 2,3-dioxygenase and prostaglandin E2 represent key mediators of the MSC-induced inhibition of NK cells. View Publication

The histo-blood group i and I antigens have been characterized as straight and branched repeats of N-acetyllactosamine,respectively,and the conversion of the straight-chain i to the branched-chain I structure on red cells is regulated to occur after birth. It has been demonstrated that the human I locus expresses 3 IGnT transcripts,IGnTA,IGnTB,and IGnTC,and that the last of these is responsible for the I branching formation on red cells. In the present investigation,the K-562 cell line was used as a model to show that the i-to-I transition in erythroid differentiation is determined by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha),which enhances transcription of the IGnTC gene,consequently leading to formation of the I antigen. Further investigation suggested that C/EBPalpha IGnTC-activation activity is modulated at a posttranslational level,and that the phosphorylation status of C/EBPalpha may have a crucial effect. Results from studies using adult and cord erythropoietic cells agreed with those derived using the K-562 cell model,with lentiviral expression of C/EBPalpha in CD34(+) hemopoietic cells demonstrating the determining role of C/EBPalpha in the induction of the IGnTC gene as well as in I antigen expression. View Publication

Mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment and are able to maintain the self-renewal capacity of hematopoietic progenitor cells (HPC). Isolation procedures for MSC vary extensively,and this may influence their biologic properties. In this study,we have compared human MSC isolated from bone marrow (BM) using two culture conditions,from cord blood (CB),and from adipose tissue (AT). The ability to maintain long-term culture-initiating cell frequency and a primitive CD34(+)CD38(-) immunophenotype was significantly higher for MSC derived from BM and CB compared with those from AT. These results were in line with a significantly higher adhesion of HPC to MSC from BM and CB versus MSC from AT. We have compared the cytokine production of MSC by cytokine antibody arrays,enzyme-linked immunosorbent assay,and a cytometric bead array. There were reproducible differences in the chemokine secretion profiles of various MSC preparations,but there was no clear concordance with differences in their potential to maintain primitive function of HPC. Global gene expression profiles of MSC preparations were analyzed and showed that adhesion proteins including cadherin-11,N-cadherin,vascular cell adhesion molecule 1,neural cell adhesion molecule 1,and integrins were highly expressed in MSC preparations derived from BM and CB. Thus,MSC from BM and CB are superior to MSC from AT for maintenance of primitive HPC. The latter property is associated with specific molecular profiles indicating the significance of cell-cell junctions but not with secretory profiles. Disclosure of potential conflicts of interest is found at the end of this article. View Publication