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

Angelman syndrome is a neurodevelopmental disorder caused by (epi)genetic lesions of maternal UBE3A. Research has focused largely on the role of UBE3A in neurons due to its imprinting in that cell type. Yet,evidence suggests there may be broader neurodevelopmental impacts of UBE3A dysregulation. Human cerebral organoids might reveal these understudied aspects of UBE3A as they recapitulate diverse cell types of the developing human brain. In this study,scRNAseq on organoids reveals the effects of UBE3A disruption on cell type-specific compositions and transcriptomic alterations. In the absence of UBE3A,progenitor proliferation and structures are disrupted while organoid composition shifts away from proliferative cell types. We observe impacts on non-neuronal cells,including choroid plexus enrichment. Furthermore,EMX1+ cortical progenitors are negatively impacted; potentially disrupting corticogenesis and delaying excitatory neuron maturation. This work reveals impacts of UBE3A on understudied cell types and related neurodevelopmental processes and elucidates potential therapeutic targets. Human cerebral organoids exhibit compositional and transcriptomic alterations in both neuronal and non-neuronal cells in the absence of UBE3A. View Publication

Galectin-1 is implicated in several pro-tumourigenic mechanisms and is considered immune-suppressive. The pharmacological inhibition of galectin-1 may be beneficial in cancers in which galectin-1 is overexpressed and driving cancer progression. This study aimed to further characterise the immunosuppressive cytokines influenced by galectin-1 in in vitro immune cell cultures and an in vivo inflammatory model using a recently discovered selective inhibitor of galectin-1,GB1908. To enable a translational approach and link mouse and human pharmacology,anti-CD3/anti-CD28 stimulated T cells cultured from human whole blood and mouse spleens were compared. For in vivo studies of T cell-mediated inflammation,the concanavalin-A (Con-A) mouse model was used to induce a T lymphocyte-driven acute liver injury phenotype. The inhibition of galectin-1 with GB1908 reduced IL-17A,IFNγ and TNFα in a concentration-dependent manner in both mouse and human T cells in vitro. The immunosuppressive cytokines measured in Con-A-treated mice were all upregulated compared to naïve mice. Subsequently,mice treated with GB1908 demonstrated a significant reduction in IL-17A,IFNγ,IL-6 and TNFα compared to vehicle-treated mice. In conclusion,galectin-1 induced the production of several important immune-suppressive cytokines from T cells in vitro and in vivo. This result suggests that,in the context of cancer therapy,a selective galectin-1 could be a viable approach as a monotherapy,or in combination with chemotherapeutic agents and/or checkpoint inhibitors,to enhance the numbers and activity of cytotoxic T cells in the tumour microenvironment of high galectin-1 expressing cancers. View Publication

Peroxisome proliferator-activated receptor gamma (PPARG) is a nuclear receptor family transcription factor (TF) critical for adipogenesis,lipid metabolism,insulin sensitivity,and inflammation. It has also been known to play essential roles in trophoblast development and placentation. Dysregulation of PPARG in trophoblast differentiation has been implicated in pregnancy complications,such as pre-eclampsia and gestational diabetes. However,the molecular mechanisms of PPARG-dependent target gene regulation and its interactions with other regulatory factors during human trophoblast differentiation remain unclear. Using human trophoblast stem cells (TSCs),mimicking placental cytotrophoblasts (CTs),and their differentiation into extravillous trophoblasts (EVTs) as our models,we reveal that PPARG has cell-type-specific targets in TSCs and EVTs. We also find that while PPARG is essential for both TSC self-renewal and EVT differentiation,only its role in EVT differentiation is ligand sensitive and requires ligand-binding domain (LBD)-mediated transcriptional activity,whereas its function in TSC self-renewal appears to be ligand insensitive. Combined analysis with chromosomal targets of previously defined key TFs in TSCs and EVTs shows that PPARG forms trophoblast cell-type-specific regulatory circuitries,leading to differential target gene regulation via transcriptional re-wiring during EVT differentiation. Additionally,the enhanced invasiveness of EVTs treated with a PPARG agonist suggests a potential connection between PPARG pathways and human placenta accreta. View Publication

BACKGROUND/OBJECTIVE A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects. MATERIALS AND METHODS Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion,the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR. RESULTS There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media,and markers such as NG2 and $\alpha$SMA in mesenchymal media. However,analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population. CONCLUSIONS CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However,nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease. 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

PURPOSE: To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM).backslashnbackslashnMETHODS: Sixty-nine rats (38 male,31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1,6,and 12 months after surgery. Both ocular tissues and systemic organs (brain,liver,kidneys,spleen,heart,and lungs) were fixed in 4% paraformaldehyde,embedded in paraffin,and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker),anti-TRA-1-85 (human cell marker),anti-Ki67 (proliferation marker),anti-CD68 (macrophage),and anti-cytokeratin (epithelial marker).backslashnbackslashnRESULTS: The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P textless 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group.backslashnbackslashnCONCLUSIONS: hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects. View Publication

Breast Cancer Stem (BCS) cells play critical roles in self-renewal,Multi Drug Resistance (MDR),differentiation and generation of secondary tumors. Conventional chemotherapy may efficiently kill the bulk of differentiated drug sensitive breast cancer cells,but not the MDR self-renewable BCS cells,leading to enrichment of the MDR BCS cells. In order to target the MDR BCS cells,we have isolated: 1) BCS cells from either breast cancer cell lines or fresh breast cancer specimens; 2) ATP binding cassette (ABC) transporter group G number 2 (ABCG2)-specific aptamers; and 3) BCS cell-binding aptamers. Interestingly,ABCG2-specific aptamers labeled the membrane surface of the ABCG2-expressing baby hamster kidney (BHK) cells,but stained whole cells of the BCS cells derived from mammospheres,implying that BCS cells might have much higher rate of endocytosis than the ABCG2-expressing BHK cells. In addition,5D3,a monoclonal antibody that recognizes the extracellular loops of ABCG2 protein,also stained whole BCS cells. Furthermore,BCS cell-binding aptamers stained whole BCS cells,but not the differentiated breast cancer MCF-7 cells. All these results support above conclusion that BCS cells might have high rate of endocytosis. Further experiments performed with aptamers and human transferrin or lactosylceramide showed that BCS cells do have much higher endocytosis rate than the differentiated breast cancer cells. Interestingly,clathrin dependent endocytosis inhibitors,such as monodansylcadaverine or sucrose,or caveolin-dependent endocytosis inhibitors,such as methyl-$$-cyclodextrin or genistein,can inhibit the internalization of transferrin or lactosylceramide into the differentiated breast cancer cells,but cannot block the internalization of these compounds into the BCS cells,suggesting that BCS cells undergo clathrin-independent and caveolin-independent endocytosis. Taken together,our data suggest that BCS cells have high rate of endocytosis and open the possibilities for delivering therapeutic agents directly into the MDR BCS cells with aptamer-coated liposomes. View Publication

BACKGROUND: The dose-limiting toxicity of doxorubicin on hematopoietic stem cells reduces the maximum benefit from this powerful drug. Melatonin may play a role in reducing this toxicity. MATERIALS AND METHODS: Melatonin at 10 microM was used while challenging human peripheral blood stem cells (PBSC) with doxorubicin (0.6 microM and 1 microM),and colony formation was used to evaluate the protective effect of melatonin. RESULTS: Melatonin was protective for the myeloid and erythroid series when given during or 1 hour after,but not before,doxorubicin,as measured by colony assay. This protection was independent from its antioxidant function as measured by 2',7'-dichlodihydro-fluorescein diacetate and was selective for PBSC when compared to the MCF-7 cancer cell line. CONCLUSION: The results suggest the importance of the time sequence for melatonin administration to exert its protective effect in relation to doxorubicin treatment,as well as its protective effect on both erythroid and myeloid elements independent from its antioxidant function. 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

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