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

FMS-related tyrosine kinase 3 ligand (FLT3L),encoded by FLT3LG,is a hematopoietic factor essential for the development of natural killer (NK),B cells,and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant,with a history of various recurrent infections,including severe cutaneous warts. The patients’ bone marrow was hypoplastic,with low levels of hematopoietic progenitors,particularly myeloid and B-cell precursors. Counts of B cells,monocytes,and DCs were low in the patients’ blood,whereas the other blood subsets,including NK cells,were affected only moderately,if at all. The patients had normal counts of Langerhans cells and dermal macrophages in the skin but lacked dermal DCs. Thus,FLT3L is required for B-cell and DC development in mice and humans. However,unlike its murine counterpart,human FLT3L is required for the development of monocytes but not NK cells. View Publication

Human embryonic stem cells (hESC) are difficult to adapt to 96-well plate assays,such as the MTT assay,because they survive best when plated as colonies,which are not easily counted and plated accurately. Two methods were developed to address this problem. In the first,ROCK inhibitor (ROCKi) was used,which allows accurate counting and plating of single hESC. In the second,small colonies were plated without ROCKi but with adaptations for accurate counting and plating. The MTT assay was also adapted for use with mouse neural stem cells. These methods allow the MTT assay to be conducted rapidly and accurately with high reproducibility between replicate experiments. When screening volatile chemicals in a 96-well plate,vapor effects may occur and dose ranges must be carefully defined. The methods were validated using the NIH assay guidance tool. These methodss could readily be translated to other 96-well plate assay. View Publication

Because neurons are difficult to obtain from humans,generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells,we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation,glutamate receptor function,and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2,resulting in high responsiveness of neural cells to glutamate,N-methyl-d-aspartate (NMDA),α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA),and (S)-3,5-d... View Publication

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However,the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study,we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette,we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly,more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification,and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional,as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore,hESCs stably modified with the CD1d gene may serve as a convenient,unlimited,and competent DC source for iNKT cell-based cancer immunotherapy. View Publication

Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX),the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene ( LRPPRC ). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions,tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study,we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC,homozygous for the LRPPRC *354V allele,and from a control,homozygous for the LRPPRC *A354 allele. Specifically,for both of these fibroblast lines we generated hiPSC,hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines,as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells,with a reduction ranging from ∼70% in hiPSC-CMs to undetectable levels in hiPSC-HLC,reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts,this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC. View Publication

The levels of transcription factor Ets1 are high in resting B and T cells,but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells,Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response,stringent control of Ets1 gene expression levels is required for homeostasis. However,the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100 kb upstream and into the gene body. Additionally,we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs,we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells,but variegated expression in T cells,suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells,we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly,functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of Ets1. View Publication

In bone marrow,cell numbers are balanced between production and loss. After chemotherapy,blood cell counts decrease initially but later recover as hematopoietic progenitor cells expand,although the mechanisms underlying this recovery are still unclear. We investigated the influence of red blood cells (RBCs) on hematopoietic stem cell (HSC) function during bone marrow recovery. Following chemotherapy,RBC concentrations in bone marrow peaked on day 5 posttreatment,coinciding with the recovery of hematopoiesis. Coculture of HSCs with RBCs resulted in a significant increase in hematopoiesis. Direct contact between RBCs and HSCs was essential for enhancement of hematopoiesis,and HSCs precultured with RBCs resulted in greater numbers of donor‐derived mature hematopoietic cells after transplantation. RNA‐sequencing analysis showed that Hes1 was the most significantly upregulated transcription factor in RBC coculture,and the response to RBC‐induced hematopoiesis of Hes1‐deficient HSCs was reduced. These findings imply a role of RBCs and Hes1 in the enhancement of hematopoietic recovery following bone marrow stress. View Publication

We studied a new method of treatment of amyotrophic lateral sclerosis with autologous mesenchymal stem cells. Autologous mesenchymal stem cells were injected intravenously (intact cells) or via lumbar puncture (cells committed to neuronal differentiation). Evaluation of the results of cell therapy after 12-month follow-up revealed slowing down of the disease progression in 10 patients in comparison with the control group consisting of 15 patients. The cell therapy was safe for the patients. View Publication

Human embryonic stem cells (hESC) have a unique capacity to self-renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied,the role of cytoplasmic regulators is still poorly characterized. Here,we report a new stem cell-specific RNA-binding protein L1TD1 (ECAT11,FLJ10884) required for hESC self-renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furthermore,we demonstrate that OCT4,SOX2,and NANOG all bind to the promoter of L1TD1. Moreover,L1TD1 is highly expressed in seminomas,and depletion of L1TD1 in these cancer cells influences self-renewal and proliferation. We show that L1TD1 colocalizes and interacts with LIN28 via RNA and directly with RNA helicase A (RHA). LIN28 has been reported to regulate translation of OCT4 in complex with RHA. Thus,we hypothesize that L1TD1 is part of the L1TD1-RHA-LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness. View Publication

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study,UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin,flt3 ligand,and granulocyte-colony stimulating factor. By week 4-5,we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor,human vascular cell adhesion molecule-1,human intracellular adhesion molecule-1,human CD31,E-selectin,and human macrophage. Furthermore,when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer,better expansion of total nucleated cells,CD34(+) cells,and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells,which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells,we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method,one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors,establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering. View Publication

An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA,they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 x 10(4) to 8.2 x 10(5) CFU/ml of cell suspension,and those for the C. coli strains ranged from 1.4 x 10(5) to 4.6 x 10(6) CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens,suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent,and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens. View Publication