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

Although studies have identified the presence of gut-associated cells in the enthesis of joints affected by spondylarthritis (SpA),a direct link through cellular transit between the gut and joint has yet to be formally demonstrated. Using KikGR transgenic mice to label in situ and track cellular trafficking from the distal colon to the joint under inflammatory conditions of both the gut and joint,we demonstrate bona-fide gut-joint trafficking of T cells from the colon epithelium,also called intraepithelial lymphocytes (IELs),to distal sites including joint enthesis,the pathogenic site of SpA. Similar to patients with SpA,colon IELs from the TNF$\Delta$ARE/+ mouse model of inflammatory bowel disease and SpA display heightened TNF production upon stimulation. Using ex vivo stimulation of photo-labeled gut-joint trafficked T cells from the popliteal lymph nodes of KikGR and KikGR TNF$\Delta$ARE/+ we saw that the CD4+ photo-labeled population was highly enriched for IL-17 competence in healthy as well as arthritic mice,however in the TNF$\Delta$ARE/+ mice these cells were additionally enriched for TNF. Using transfer of magnetically isolated IELs from TNF+/+ and TNF$\Delta$ARE/+ donors into Rag1 -/- hosts,we confirmed that IELs can exacerbate inflammatory processes in the joint. Finally,we blocked IEL recruitment to the colon epithelium using broad spectrum antibiotics in TNF$\Delta$ARE/+ mice. Antibiotic-treated mice had reduced gut-joint IEL migration,contained fewer Il-17A and TNF competent CD4+ T cells,and lessened joint pathology compared to untreated littermate controls. Together these results demonstrate that pro-inflammatory colon-derived IELs can exacerbate inflammatory responses in the joint through systemic trafficking,and that interference with this process through gut-targeted approaches has therapeutic potential in SpA. View Publication

BACKGROUND Friedreich's ataxia (FRDA),a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy,is caused by silencing of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. METHODS Application of our previously established FRDA human induced pluripotent stem cell (hiPSC) derived cardiomyocytes model as a platform to assess the efficacy of treatment with either the antioxidant coenzyme Q10 analog,idebenone (IDE) or the iron chelator,deferiprone (DFP),which are both under clinical trial. RESULTS DFP was able to more significantly suppress synthesis of reactive oxygen species (ROS) than IDE at the dosages of 25 $\$ and 10nM respectively which agreed with the reduced rate of intracellular accumulation of iron by DFP treatment from 25 to 50 $\$ With regard to cardiac electrical-contraction (EC) coupling function,decay velocity of calcium handling kinetics in FRDA-hiPSC-cardiomyocytes was significantly improved by DFP treatment but not by IDE. Further mechanistic studies revealed that DFP also modulated iron induced mitochondrial stress as reflected by mitochondria network disorganization and decline level of respiratory chain protein,succinate dehydrogenase (CxII) and cytochrome c oxidase (COXIV). In addition,iron-response protein (IRP-1) regulatory loop was overridden by DFP as reflected by resumed level of ferritin (FTH) back to basal level and the attenuated transferrin receptor (TSFR) mRNA level suppression thereby reducing further iron uptake. CONCLUSIONS DFP modulated iron homeostasis in FRDA-hiPSC-cardiomyocytes and effectively relieved stress-stimulation related to cardiomyopathy. The resuming of redox condition led to the significantly improved cardiac prime events,cardiac electrical-coupling during contraction. View Publication

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study,we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPARγ2,lipoprotein lipase,and adipsin increased during adipocytogenesis of hMSCs. Simultaneously,the expression of canonical WNT2,10B,13,and 14 decreased,whereas non-canonical WNT4 and 11 increased,and WNT5A was unchanged. A small molecule WNT mimetic,SB-216763,increased accumulation of β-catenin protein,inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast,knockdown of β-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells. View Publication

BACKGROUND AIMS: Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently,stem cell culture has been carried out using feeder cells,and culture media,that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells,essential conditions for the use of stem cells for clinical purposes. To date,however,there has been limited success in achieving this aim. METHODS,RESULTS AND CONCLUSIONS: Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntington's disease and myotonic dystrophy 1) genomically stable hES cell lines,and their adaptation to feeder-free culture,all under xeno-free conditions. View Publication

The mammary gland undergoes significant remodeling during pregnancy and lactation,which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self-renewal in breast cancer. Here,we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4,SOX2,NANOG,known to constitute the core self-renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts,a population of hBSCs exhibited an encapsulated ESC-like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self-renewal TFs were found silenced in the normal resting epithelium,they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore,hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient-specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer,with potential use in bioengineering and tissue regeneration. View Publication

Nuclear reprogramming of adult somatic tissue enables embryo-independent generation of autologous,patient-specific induced pluripotent stem (iPS) cells. Exploiting this emergent regenerative platform for individualized medicine applications requires the establishment of bioequivalence criteria across derived pluripotent lines and lineage-specified derivatives. Here,from individual patients with type 1 diabetes (T1D) multiple human iPS clones were produced and prospectively screened using a battery of developmental markers to assess respective differentiation propensity and proficiency in yielding functional insulin (INS)-producing progeny. Global gene expression profiles,pluripotency expression patterns,and the capacity to differentiate into SOX17- and FOXA2-positive definitive endoderm (DE)-like cells were comparable among individual iPS clones. However,notable intrapatient variation was evident upon further guided differentiation into HNF4α- and HNF1β-expressing primitive gut tube,and INS- and glucagon (GCG)-expressing islet-like cells. Differential dynamics of pluripotency-associated genes and pancreatic lineage-specifying genes underlined clonal variance. Successful generation of glucose-responsive INS-producing cells required silencing of stemness programs as well as the induction of stage-specific pancreatic transcription factors. Thus,comprehensive fingerprinting of individual clones is mandatory to secure homogenous pools amenable for diagnostic and therapeutic applications of iPS cells from patients with T1D. View Publication

BACKGROUND Various markers are used to identify the unique sub-population of breast cancer cells with stem cell properties. Whether these markers are expressed in all breast cancers,identify the same population of cells,or equate to therapeutic response is controversial. METHODS We investigated the expression of multiple cancer stem cell markers in human breast cancer samples and cell lines in vitro and in vivo,comparing across and within samples and relating expression with growth and therapeutic response to doxorubicin,docetaxol and radiotherapy. RESULTS CD24,CD44,ALDH and SOX2 expression,the ability to form mammospheres and side-population cells are variably present in human cancers and cell lines. Each marker identifies a unique rather than common population of cancer cells. In vivo,cells expressing these markers are not specifically localized to the presumptive stem cell niche at the tumour/stroma interface. Repeated therapy does not consistently enrich cells expressing these markers,although ER-negative cells accumulate. CONCLUSIONS Commonly employed methods identify different cancer cell sub-populations with no consistent therapeutic implications,rather than a single population of cells. The relationships of breast cancer stem cells to clinical parameters will require identification of specific markers or panels for the individual cancer. View Publication

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However,limited information is available regarding the immunologic features of iPSCs. In this study,expression of MHC and T cell co-stimulatory molecules in hiPSCs,and the effects on activation,proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation,hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ,TNF-α and IL-17,hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10,and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity,which may result from their induction of IL-10-secreting Treg. View Publication

An investigation was conducted to demonstrate that neurodazine (Nz) and neurodazole (Nzl),two imidazole-based small molecules,promote neuronal differentiation in both neuroblastoma and fibroblast cells. The results show that differentiated cells generated by treatment with Nz and Nzl express neuron-specific markers. The ability of Nz and Nzl to induce neurogenesis of neuroblastoma and fibroblast cells was found to be comparable to those of the known neurogenic factors,retinoic acid and trichostatin A. In addition,the cells differentiated by Nz and Nzl are observed to express different isoforms of glutamate receptors. The results of signaling pathway studies reveal that two substances enhance neurogenesis in neuroblastoma cells by activating Wnt and Shh signaling pathways and neurogenesis in fibroblast cells by mainly activating the Wnt signaling pathway. Observations made in the present study suggest that Nz and Nzl will serve as chemical tools to generate specific populations of neuronal cells from readily available and simply manageable cells. View Publication

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC) show great promise as autologous donor cells to treat heart disease. A major technical obstacle to this approach is that available induction methods often produce heterogeneous cell population with low percentage of cardiomyocytes. Here we describe a cardiac enrichment approach using non-integrating adeno-associated virus (AAV). We first examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes. Result showed that AAV1 demonstrated the highest in vitro transduction efficiency among seven widely used serotypes. Next differentiated iPSC derivatives were transduced with drug-selectable AAV1 expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to 57% in two weeks. Compared to other enrichment strategies such as integrative genetic selection,mitochondria labeling or surface marker cell sorting,this simple AAV method described herein bypasses antibody or dye labeling. These findings provide proof-of-concept for large-scale cardiomyocyte enrichment by exploiting AAV's intrinsic tissue tropism. View Publication

Patient safety is a major concern in the application of induced pluripotent stem cells (iPSCs) in cell-based therapy. Efforts are being made to reprogram,maintain,and differentiate iPSCs in defined conditions to provide a safe source of stem cells for regenerative medicine. Recently,human fibroblasts were successfully reprogrammed into pluripotent stem cells using four recombinant proteins (OCT4,c-Myc,KLF4,and SOX2) fused with a cell-penetrating peptide (9R). These protein-induced pluripotent stem cells (piPSCs) are maintained and propagated on a feeder layer of mouse embryonic fibroblasts. Use of animal-derived products in maintenance and differentiation of iPSCs poses risks of zoonotic disease transmission and immune rejection when transplanted into humans. To avoid potential incorporation of xenogenic products,we cultured piPSCs on recombinant human matrix proteins. We then tested whether recombinant human matrix proteins can support self-renewal and pluripotency of piPSCs. After long-term culture on recombinant human vitronectin in xeno-free conditions,piPSCs retained the expression of pluripotent markers. The pluripotency of these cells was further evaluated by differentiating toward ectoderm,mesoderm,and endoderm lineages in vitro. In conclusion,recombinant human vitronectin can support the long-term culture and maintain the stemness of piPSCs in defined nonxenogenic conditions. View Publication