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

Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver,very few HSCs efflux Hoechst 33342 efficiently,and they are thus not detected as side population" (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment� View Publication

OBJECTIVE: Endothelial-like vascular progenitor cells (VPCs) can be collected in peripheral blood stem cell (PBSC) products that are used in hematopoietic stem cell transplantation (HSCT). The association between VPCs in PBSC products and transplant-related toxicity caused by high-dose chemo/radiotherapy was assessed to identify potential mediators of vascular repair. MATERIALS AND METHODS: PBSC grafts in 29 patients (mean age: 48 years; range,20-67 years) undergoing autologous HSCT were analyzed using a cell culture assay for VPC cluster formation in fibronectin-coated dishes in serum-rich angiogenic conditions. Transplant toxicity was estimated using total length of hospital stay (LOS) following HSCT and the Seattle criteria for transplant-related organ toxicity for 8 organ systems (grade 0-4). RESULTS: LOS following graft reinfusion was lower (14.7 vs 20.0 days,p = 0.002) and the mean number of organs with any toxicity (1.0 vs 2.4,p = 0.016) or with toxicity grade textgreater or = 2 was reduced (0.2 vs 1.6 organs,p = 0.007) in patients with high graft VPC content (n = 10,textgreater2.0 x 10(3) VPCs/kg) compared with reduced VPC content (n = 19,textless or = 2.0 x 10(3) VPCs/kg). An association between graft CD34(+) levels and LOS or organ toxicity was not observed. In addition,graft VPC levels were independent of graft CD34 counts,peripheral blood monocytes and hemoglobin levels,age,and disease (p = NS). CONCLUSION: PBSC products enriched for VPCs are associated with reduced toxicity following HSCT. Identifying specific factors that contribute to high graft VPC levels is needed. View Publication

The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare,naturally occurring,homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here,we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human,mobilized hematopoietic stem progenitor cells (HSPC),resulting in a transplant that undergoes normal hematopoiesis,produces CCR5 null T cells,and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection. Subject terms: HIV infections,CRISPR-Cas9 genome editing,Retrovirus,Translational research View Publication

BAG-1 protein has been well characterized as necessary for proper neuronal development. However,little is known about the function of BAG-1 in the adult brain. In this work,the expression and localization of BAG-1 in the mature mouse brain was studied. The levels of both BAG-1 isoforms decrease significantly in the brain during development. BAG-1 was found preferentially expressed in Neuronal Precursor Cells (NPCs) in the two major niches of neurogenesis. Lentiviral mediated overexpression of BAG-1 increased the proliferation rate of cultured NPCs. In addition,depletion of BAG-1 from NPCs induced a decrease in NPCs proliferation in the presence of a stress hormone,corticosterone. These data suggest a role for BAG-1 in mechanisms of neurogenesis in the adult mouse brain. View Publication

The possibility of using cell-based therapeutics to treat cardiac failure has generated significant interest since the initial introduction of stem cell-based technologies. However,the methods to quickly and robustly direct stem cell differentiation towards cardiac cell types have been limited by a reliance on recombinant growth factors to provide necessary biological cues. We report here the use of dorsomorphin homologue 1 (DMH1),a second-generation small molecule BMP inhibitor based on dorsomorphin,to efficiently induce beating cardiomyocyte formation in mouse embryonic stem cells (ESCs) and to specifically upregulate canonical transcriptional markers associated with cardiac development. DMH1 differs significantly from its predecessor by its ability to enrich for pro-cardiac progenitor cells that respond to late-stage Wnt inhibition using XAV939 and produce secondary beating cardiomyocytes. Our study demonstrates the utility of small molecules to complement existing in vitro cardiac differentiation protocols and highlights the role of transient BMP inhibition in cardiomyogenesis. View Publication

BACKGROUND Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10{\%} DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry,as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel,each sample was flow sorted into fibroblast,T-cell,B-cell,and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS Upon dissociation,cryopreserved synovial tissue fragments yielded a high frequency of viable cells,comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with {\~{}} 30 arthroplasty and {\~{}} 20 biopsy samples yielded a consensus digestion protocol using 100 mu$g/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes,distinct populations of memory B cells and antibody-secreting cells,and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes,fibroblasts,and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell,including transcripts encoding characteristic lineage markers identified. CONCLUSIONS We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers. View Publication

Experimental evidence indicates that shear stress (SS) exerts a morphogenetic function during cardiac development of mouse and zebrafish embryos. However,the molecular basis for this effect is still elusive. Our previous work described that in adult endothelial cells,SS regulates gene expression by inducing epigenetic modification of histones and activation of transcription complexes bearing acetyltransferase activity. In this study,we evaluated whether SS treatment could epigenetically modify histones and influence cell differentiation in mouse embryonic stem (ES) cells. Cells were exposed to a laminar SS of 10 dyne per cm2/s(-1),or kept in static conditions in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). These experiments revealed that SS enhanced lysine acetylation of histone H3 at position 14 (K14),as well as serine phosphorylation at position 10 (S10) and lysine methylation at position 79 (K79),and cooperated with TSA,inducing acetylation of histone H4 and phosphoacetylation of S10 and K14 of histone H3. In addition,ES cells exposed to SS strongly activated transcription from the vascular endothelial growth factor (VEGF) receptor 2 promoter. This effect was paralleled by an early induction of cardiovascular markers,including smooth muscle actin,smooth muscle protein 22-alpha,platelet-endothelial cell adhesion molecule-1,VEGF receptor 2,myocyte enhancer factor-2C (MEF2C),and alpha-sarcomeric actin. In this condition,transcription factors MEF2C and Sma/MAD homolog protein 4 could be isolated from SS-treated ES cells complexed with the cAMP response element-binding protein acetyltransferase. These results provide molecular basis for the SS-dependent cardiovascular commitment of mouse ES cells and suggest that laminar flow may be successfully applied for the in vitro production of cardiovascular precursors. View Publication

Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However,only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8-week-old mice were dissociated and cultured to allow sphere colony formation. Wnt3a,a glycogen synthase kinase (GSK) 3 inhibitor,fibroblast growth factor (FGF) 2,and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki-67-positive proliferating cells in monolayer culture. The Wnt3a-treated primary sphere cells were capable of self-renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a-treated cells showed nuclear accumulation of beta-catenin,and a GSK3 inhibitor,SB216763,mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2,with Ki-67-positive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells,and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells. View Publication

The epigenetics of early commitment to embryonal cardiomyocyte is poorly understood. In this work,we compared the effect of thyroid hormone and that of anacardic acid,a naturally occurring histone acetylase inhibitor,or both in combination,on mouse embryonic stem cells (mES) differentiating into embryonal cardiomyocyte by embryoid bodies (EBs) formation. Although the results indicated that anacardic acid (AA) and thyroid hormone were both efficient in promoting cardiomyocyte differentiation,we noticed that a transient exposure of mES to AA alone was sufficient to enlarge the beating areas of EBs compared to those of untreated controls. This effect was associated with changes in the chromatin structure at the promoters of specific cardiomyogenic genes. Among them,a rapid induction of the transcription factor Castor 1 (CASZ1),important for cardiomyocytes differentiation and maturation during embryonic development,was observed in the presence of AA. In contrast,thyroid hormone (T 3) was more effective in stimulating spontaneous firing,thus suggesting a role in the production of a population of cardiomyocyte with pacemaker properties. In conclusion,AA and thyroid hormone both enhanced cardiomyocyte formation along in apparently distinct pathways. View Publication

Since the discovery of neural stem cells (NSC) in the embryonic and adult mammalian central nervous system (CNS),there have been a growing numbers of tissue culture media and protocols to study and functionally characterize NSCs and its progeny in vitro. One of these culture systems introduced in 1992 is referred to as the Neurosphere Assay,and it has been widely used to isolate,expand,differentiate and even quantify NSC populations. Several years later because its application as a quantitative in vitro assay for measuring NSC frequency was limited,a new single-step semisolid based assay,the Neural Colony Forming Cell (NCFC) assay was developed to accurately measure NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e.,their proliferative potential). The evolution and continued improvements made to these tissue culture tools will facilitate further advances in the promising application of NSCs for therapeutic use. View Publication

Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7,P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture,but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive,glucose-transporter-2-low (Ins(+)Glut2(LO)) cells,representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7,were retained into adulthood,and a subset differentiated into endocrine,ductal,and neural lineages,illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new,functional β-cells,and which may be potentially exploited for regenerative therapies in the future. View Publication

Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However,an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study,we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1,side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore,knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice,supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion,but not for CSC maintenance. Taken together,our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus,targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer. View Publication