搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Human osteogenic progenitors are not precisely defined,being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells (MSCs). Notably,select human pericytes can develop into bone-forming osteoblasts. Here,we sought to define the differentiation potential of CD146+ human pericytes from skeletal and soft tissue sources,with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte. CD146+CD31-CD45- pericytes were derived by fluorescence-activated cell sorting from human periosteum,adipose,or dermal tissue. Periosteal CD146+CD31-CD45- cells retained canonical features of pericytes/MSC. Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo,while soft tissue pericytes did not readily. Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts,and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes. Conversely,enrichment of CXCR4+ pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell. In sum,human skeletal and soft tissue pericytes differ in their basal abilities to form bone. Diversity exists in soft tissue pericytes,however,and CXCR4+ pericytes represent an osteoblastogenic,non-adipocytic cell precursor. Indeed,enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering. View Publication

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions,wherein the regulation of Wnt/$\beta$-catenin pathway is often desirable. Here,we examined the effect of trolox,a vitamin E analog,on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/$\beta$-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/$\beta$-catenin regulator. View Publication

The airway epithelium contains ionocytes,a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR),a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high ({\~{}}3{\%}) ionocyte abundance in ex vivo nasal samples,with no difference between CF and control individuals. In bronchi,ionocytes instead appeared very rarely as previously reported,thus suggesting a possible proximal-distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture,which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure,we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions,thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern. View Publication

Management of chronic kidney disease (CKD) remains a major challenge due limited therapeutic options to reverse fibrosis,which is a critical feature in CKD. Partial epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs) is a key driver of fibrosis,and has become an important focus for kidney protection strategies. Blood platelets,a major source of circulating transforming growth factor beta (TGF-β),are implicated in pathogenesis of CKD,but their involvement in EMT and kidney fibrosis remains uncertain. Methods: We used two mouse models of renal fibrosis—diabetic kidney disease (DKD) and unilateral ureter obstruction (UUO)—to examine the connection between platelets,partial EMT,and fibrosis. Platelet inhibition or depletion was performed to assess EMT,cell cycle arrest,and fibrosis. In vitro,platelets were applied to TECs and kidney organoids. To determine the role of TGF-β signaling,we used TGF-βRI inhibitor. Expression of EMT,and fibrosis markers,as well as TGF-β1 signaling,were analyzed using western blot,reverse transcription quantitative PCR (RT-qPCR),enzyme-linked immunosorbent assay (ELISA),and immunostaining. Results: In both animal models,platelet inhibition or depletion resulted in reduced expression of cell cycle arrest marker p21,partial EMT and fibrosis. In vitro,activated platelets stimulated cell cycle arrest,EMT,and fibrosis in TECs and kidney organoids. Chronically injured TECs experience cell-cycle arrest which promote a paracrine EMT program in TECs,jointly leading to fibrosis. This platelet-mediated effect on cell cycle arrest and EMT was driven by TGF-β1 signaling,as selective inhibition of the TGF-β receptor rescued these dysfunctional phenotypes. Conclusions: Our study demonstrates that platelets activate the TGF-β1 pathway,leading to cell cycle arrest,EMT and renal fibrosis. These findings suggest that antiplatelet therapies may have potential renoprotective effects by protecting tubular homeostasis,attenuating partial EMT and fibrosis. View Publication

Induced pluripotent stem cells (iPSCs) are commonly used to model human genetic diseases. Two main strategies are used. The first involves making iPSC lines from individual cases with a disease,and the second involves making disease-relevant gene edits in established iPSC lines. Because generating gene-edited lines is time consuming and expensive,most studies begin with one starting iPSC stock line and evaluate several gene-edited sublines. The current studies focus on gene-editing to model Lesch–Nyhan disease (LND),which is caused by mutations in the HPRT1 gene. The same pathogenic c.508C>T edit was made in four well-established stock lines,and three gene-edited lines were isolated from each. RNA sequencing (RNAseq) was,then,used to evaluate the impact of the gene edit. Gene-edited lines were compared to their corresponding stock lines,as well as to each other. An aggregate analysis of all lines combined was also conducted to determine the most robust findings across all lines. Results from gene editing were further compared with iPSC lines derived from individual cases with LND,to determine how closely findings from gene editing match results obtained with case-derived lines. There were two main findings. First,the same gene edit has a different impact on gene expression when starting with different starting stock lines. Second,the gene editing strategy does not produce the same results as the case-derived strategy. Potential explanations for these differences are addressed,along with the relevance of these two different strategies for disease modeling. View Publication

The transcription factor NF-kappaB has anti-apoptotic properties and may confer chemoresistance to cancer cells. Here,we describe human pancreatic carcinoma cell lines that differ in the responsiveness to the topoisomerase-2 inhibitors VP16 (20 microM) and doxorubicin (0.3 microM): Highly sensitive T3M4 [corrected] and PT45-P1 cells,and Capan-1 and A818-4 cells that were almost resistant to both anti cancer drugs. VP16,but not doxorubicin,transiently induced NF-kappaB activity in all cell lines,whereas basal NF-kappaB binding was nearly undetectable in T3M4 [corrected] and PT45-P1 cells,but rather high in Capan-1 and A818-4 cells,as demonstrated by gel-shift and luciferase assays. Treatment with various NF-kappaB inhibitors (Gliotoxin,MG132 and Sulfasalazine),or transfection with the IkappaBalpha super-repressor,strongly enhanced the apoptotic effects of VP16 or doxorubicin on resistant Capan-1 and 818-4 cells. Our results indicate that under certain conditions the resistance of pancreatic carcinoma cells to chemotherapy is due to their constitutive NF-kappaB activity rather than the transient induction of NF-kappaB by some anti-cancer drugs. Blockade of basal NF-kappaB activity by well established drugs efficiently reduces chemoresistance of pancreatic cancer cells and offers the potential for improved therapeutic strategies. View Publication

Astrocytes are instrumental to major brain functions,including metabolic support,extracellular ion regulation,the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental,psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes),we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP,S100$\$,NFIA and ALDH1L1. In addition,mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion,the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy. View Publication

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs),but little is known about SSEA-5 expression in other primitive tissues (e.g.,human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors,cell lines,teratomas,and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors,such as embryonal carcinoma. In tumor cell lines,SSEA-5 was highly immunoreactive in Capan-1 cells,while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs,with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use. View Publication

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies,we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system,which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor,yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures,revealing that the reaction engenders both MBC subsets with different immune effector function and,ultimately,LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity,for vaccine development,and for understanding long-term pathogen resistance. View Publication

Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a mitochondrial disorder characterized by myoclonus epilepsy,generalized seizures,ataxia and myopathy. MERRF syndrome is primarily due to an A to G mutation at mtDNA 8344 that disrupts the mitochondrial gene for tRNA(Lys). However,the detailed mechanism by which this tRNA(Lys) mutation causes mitochondrial dysfunction in cardiomyocytes or neurons remains unclear. In this study,we generated human induced pluripotent stem cells (hiPSCs) that carry the A8344G genetic mutation from patients with MERRF syndrome. Compared with mutation-free isogenic hiPSCs,MERRF-specific hiPSCs (MERRF-hiPSCs) exhibited reduced oxygen consumption,elevated reactive oxygen species (ROS) production,reduced growth,and fragmented mitochondrial morphology. We sought to investigate the induction ability and mitochondrial function of cardiomyocyte-like cells differentiated from MERRF-hiPSCs. Our data demonstrate that that cardiomyocyte-like cells (MERRF-CMs) or neural progenitor cells (MERRF-NPCs) differentiated from MERRF-iPSCs also exhibited increased ROS levels and altered antioxidant gene expression. Furthermore,MERRF-CMs or -NPCs contained fragmented mitochondria,as evidenced by MitoTracker Red staining and transmission electron microscopy. Taken together,these findings showed that MERRF-hiPSCs and MERRF-CM or -NPC harboring the A8344G genetic mutation displayed contained mitochondria with an abnormal ultrastructure,produced increased ROS levels,and expressed upregulated antioxidant genes. View Publication

It has been proposed that the ancestral fungus was mating competent and homothallic. However,many mating-competent fungi were initially classified as asexual because their mating capacity was hidden behind layers of regulation. For efficient in vitro mating,the essentially obligate diploid ascomycete pathogen Candida albicans has to change its mating type locus from heterozygous MTL a /α to homozygous MTL a / a or MTL α/α and then undergo an environmentally controlled epigenetic switch to the mating-competent opaque form. These requirements greatly reduce the potential for C. albicans mating. Deletion of the Yci1 domain gene OFR1 bypasses the need for C. albicans cells to change the mating type locus from heterozygous to homozygous prior to switching to the opaque form and mating and allows homothallic mating of MTL heterozygous strains. This bypass is carbon source dependent and does not occur when cells are grown on glucose. Transcriptional profiling of ofr1 mutant cells shows that in addition to regulating cell type and mating circuitry,Ofr1 is needed for proper regulation of histone and chitin biosynthesis gene expression. It appears that OFR1 is a key regulator in C. albicans and functions in part to maintain the cryptic mating phenotype of the pathogen. View Publication