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

BACKGROUND Cardiovascular cell therapy represents a promising field,with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease") consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility� View Publication

Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However,responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here,we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction,cell-cycle progression,and transcriptional responses in HSPC subpopulations,with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation,constraining proliferation,yield,and engraftment of edited HSPCs. However,functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs. View Publication

Recently,vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However,it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here,we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted,WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery,T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells,whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells,but not the virus-specific T cells,displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells,the animals did not develop autoimmunity,and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells. View Publication

Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular,hematological,and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT(+) and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2,OCT4,KLF-4,and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72 h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2(-/-),$\$-chain(-/-),C2(-/-) immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment,immunostaining,and RT-PCR. Additionally,the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery. View Publication

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders,hence they make suitable targets in proteomic studies. However,extraction of membrane proteins is challenging due to their low abundance,stability,and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction,the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols,internally identified as Protocol 1 and 2,were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation,while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer's recommendation. Using Protocol 2,protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150-200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships,particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states,such as ME/CFS. View Publication

BACKGROUND AIMS: Previous studies have demonstrated that the combination of granulocyte-colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34(+) hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets. METHODS: We characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation,and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor. RESULTS: The number of aldehyde dehydrogenase (ALDH)(bright) and CD34(+) cells was significantly higher after plerixafor treatment (1.2-5.0 and 1.5-6.0 times; both P textless 0.01) and an enrichment of the very primitive CD34(+) CD38(-) and ALDH(bright) CD34(+) CD38(-) HSC subsets was detectable. Additionally,two distinct ALDH(+) subsets could be clearly distinguished. The small ALDH(high) subset showed a higher number of CD34(+) CD38(-) cells in contrast to the total ALDH(bright) subpopulation and probably represented a very primitive subpopulation of HSC. CONCLUSIONS: A combined staining of ALDH,CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34(+) cells but was also able to increase the proportion of more primitive stem cell subsets. View Publication

Our previous studies have shown that ethanol intoxication combined with burn injury increases intestinal bacterial growth,disrupts the intestinal barrier,and enhances bacterial translocation. Additionally,studies show that Th17 effector cytokines IL-17 and IL-22,which are dependent on IL-23,play important roles in maintaining intestine mucosal barrier integrity. Recent findings suggest neutrophils are a significant source of IL-17 and IL-22. We determined the effect of ethanol and burn injury on neutrophil IL-17 and IL-22 production,as well as their ability to phagocytose and in bacterial clearance,and whether these effects are modulated by IL-23. Mice were given ethanol 4 h prior to receiving ˆ¼12.5% total body surface area burn and were euthanized day 1 after injury. We observed that intoxication combined with burn injury significantly decreases blood neutrophil phagocytosis and bacteria killing,as well as their ability to produce IL-17 and IL-22,compared with sham vehicle mice. The treatment of neutrophils with rIL-23 significantly increases IL-22 and IL-17 release and promotes expression of IL-23R,retinoic acid-related orphan receptor $\gamma$t,Lipocalin2,and Nod-like receptor 2 following ethanol and burn injury. Furthermore,IL-22- and IL-17-producing neutrophils have enhanced neutrophil extracellular trap formation and bacterial killing ability,which are dependent on IL-23. Finally,although we observed that peritoneal neutrophils harvested after casein treatment are functionally different from blood neutrophils,both blood and peritoneal neutrophils exhibited the same response to rIL-23 treatment. Together these findings suggest that IL-23 promotes neutrophil IL-22 and IL-17 production and their ability to kill bacteria following ethanol and burn injury. View Publication

High-throughput massively parallel reporter assays (MPRAs) and phenotype-rich in vivo transgenic mouse assays are two potentially complementary ways to study the impact of noncoding variants associated with psychiatric diseases. Here,we investigate the utility of combining these assays. Specifically,we carry out an MPRA in induced human neurons on over 50,000 sequences derived from fetal neuronal ATAC-seq datasets and enhancers validated in mouse assays. We also test the impact of over 20,000 variants,including synthetic mutations and 167 common variants associated with psychiatric disorders. We find a strong and specific correlation between MPRA and mouse neuronal enhancer activity. Four out of five tested variants with significant MPRA effects affected neuronal enhancer activity in mouse embryos. Mouse assays also reveal pleiotropic variant effects that could not be observed in MPRA. Our work provides a catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays. MPRAs and in vivo transgenic mouse assays are two potentially complementary ways to assay the impact of noncoding variants. Here,authors find a strong and specific correlation between the assays in neural cells. Mouse assays also reveal pleiotropic effects not observed in MPRA. View Publication

DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway,which promotes error-free repair of DNA double-strand breaks,is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus,cells from Fanconi anemia patients,which lack this critical pathway,fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study,we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6,but not E7,recovers FA iPSC colony formation and,furthermore,that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase,NANOG,and Tra-1-60,indicating that they were fully reprogrammed into pluripotent cells. However,FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression,whereas their FA-complemented counterparts grew efficiently. Thus,we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. IMPORTANCE A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53. View Publication

Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7,which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment,behavioral abnormalities and nervous system defects,though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS,we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation,not cholesterol deficiency,is critical for SLOS-associated defects. We further identified downregulation of Wnt/$$-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs,suggesting that Wnt signaling may be a promising therapeutic target for SLOS. View Publication