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

Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here,memory B cells are activated and amplified using Epstein-Barr virus infection,co-cultured with CHO-muCD40L cells,and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells,and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly,our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family,influenza A neutralizing antibodies,contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. View Publication

Human osteogenic progenitors are not precisely defined,being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells (MSCs). Notably,select human pericytes can develop into bone-forming osteoblasts. Here,we sought to define the differentiation potential of CD146+ human pericytes from skeletal and soft tissue sources,with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte. CD146+CD31-CD45- pericytes were derived by fluorescence-activated cell sorting from human periosteum,adipose,or dermal tissue. Periosteal CD146+CD31-CD45- cells retained canonical features of pericytes/MSC. Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo,while soft tissue pericytes did not readily. Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts,and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes. Conversely,enrichment of CXCR4+ pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell. In sum,human skeletal and soft tissue pericytes differ in their basal abilities to form bone. Diversity exists in soft tissue pericytes,however,and CXCR4+ pericytes represent an osteoblastogenic,non-adipocytic cell precursor. Indeed,enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering. View Publication

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs),and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo,but is essential for normal HSC/HPC differentiation. In addition,Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover,deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and,subsequently,improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. View Publication

Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis,and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here,we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus,a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus,this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome. View Publication

SummaryBackgroundMacrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However,the production and application of genetically edited,highly effective,and mass-produced CAR-modified macrophages (CAR-Ms) are challenging.MethodsHere,we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach,together with a simple differentiation protocol,produced stable and highly effective CAR-Ms without heterogeneity.FindingsThese engineered cells phagocytosed cancer cells,leading to significant inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore,the engineered CARs,which incorporated a combination of CD3? and Megf10 (referred to as FRP5M?),markedly enhanced the antitumour effect of CAR-Ms by promoting M1,but not M2,polarisation. FRP5M? promoted M1 polarisation via nuclear factor kappa B (NF-?B),ERK,and STAT1 signalling,and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling,inducing M1 polarisation of bystander non-CAR macrophages,and enhancing the infiltration of T cells in cancer spheroids.InterpretationOur findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion,the guided insertion of CAR containing CD3? and Megf10 domains is an effective strategy for the immunotherapy of solid tumours.FundingThis work was supported by KRIBB Research Initiative Program Grant (KGM4562431,KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,10.13039/501100003625Ministry of Health and Welfare) (22A0304L1-01). View Publication

Redirecting Ag specificity by transfer of TCR genes into PBLs is an attractive method to generate large numbers of cytotoxic T cells for immunotherapy of cancer and viral diseases. However,transferred TCR chains can pair with endogenous TCR chains,resulting in the formation of mispaired TCR dimers and decreased or unspecific reactivity. TCR gene transfer into hematopoietic stem cells (HSCs) is an alternative to create T cells with desired Ag specificity,because in this case expression of endogenous TCR chains is then less likely owing to allelic exclusion. We generated TCR-transduced T cells from peripheral T cells using the lymphocytic choriomeningitis virus-specific P14 TCR. After transfer of the P14 TCR genes into HSCs and subsequent reconstitution of irradiated mice,TCR-engineered HSC-derived T cells were produced. We then compared the Ag-specific T cell populations with P14 TCR-transgenic T cells for their therapeutic efficiency in three in vivo models. In this study,we demonstrate that TCR-transduced T cells and TCR-engineered HSC-derived T cells are comparable in controlling lymphocytic choriomeningitis virus infection in mice and suppress growth of B16 tumor cells expressing the cognate Ag in a comparable manner. View Publication

Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally,antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails,but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here,we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally,we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example,H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique. View Publication