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

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment landscape of hematologic cancers by engineering T cells to specifically target and destroy cancer cells. Monitoring CAR T cell activity and function is essential for optimizing therapeutic outcomes,but existing tools for CAR detection are often limited in specificity and functional assessment capability. Methods: We developed dextran multimers by conjugating multiple CAR-specific antigens to a dextran backbone. The multimers were compared to previously reported antigen tetramers for their ability to stain and detect CAR T cells. Because these multimers incorporate the CAR target antigen,they uniquely enable assessment of CAR T cell functionality. We tested the staining and functional properties of the multimers across a range of CAR constructs with different affinities,using flow cytometry and microscopy. Results: The dextran multimers demonstrated high specificity and sensitivity in staining CAR T cells,with adjustable antigen density to optimize binding. Dextran multimers also enabled effective clustering and subsequent activation of CARs,showing their utility as both a staining and functional assessment tool. The multimers revealed that CARs with different affinities and clustering tendencies displayed varied binding and activation in response to different antigen densities. Conclusions: Dextran multimers offer a dual advantage as versatile reagents for both staining and functional analysis of CAR T cells. Their capacity to engage CARs with the specific antigen provides a valuable platform for evaluating CAR functionality,informing CAR design improvements,and enhancing therapeutic precision. View Publication

Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1 −/+ ) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1 +/+ ) HSPCs upon transplantation in mice. Based on this evidence,we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice,and in lineage specific,regulated and robust transgene expression. This approach,which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells,may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases. Subject terms: Targeted gene repair,Haematopoietic stem cells,Microglial cells View Publication

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV,purified HPCs were exposed to KSHV,and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells,and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection,persistence,and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo. View Publication

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha,we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally,enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus,the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling,and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution. View Publication

Prenatal lethality associated with mouse knockout of Mettl16,a recently identified RNA N6-methyladenosine (m 6 A) methyltransferase,has hampered characterization of the essential role of METTL16-mediated RNA m 6 A modification in early embryonic development. Here,using cross-species single-cell RNA sequencing analysis,we found that during early embryonic development,METTL16 is more highly expressed in vertebrate hematopoietic stem and progenitor cells (HSPCs) than other methyltransferases. In Mettl16-deficient zebrafish,proliferation capacity of embryonic HSPCs is compromised due to G1/S cell cycle arrest,an effect whose rescue requires Mettl16 with intact methyltransferase activity. We further identify the cell-cycle transcription factor mybl2b as a directly regulated by Mettl16-mediated m 6 A modification. Mettl16 deficiency resulted in the destabilization of mybl2b mRNA,likely due to lost binding by the m 6 A reader Igf2bp1 in vivo. Moreover,we found that the METTL16-m 6 A- MYBL2 -IGF2BP1 axis controlling G1/S progression is conserved in humans. Collectively,our findings elucidate the critical function of METTL16-mediated m 6 A modification in HSPC cell cycle progression during early embryonic development. View Publication

The activity of neural precursor cells in the adult hippocampus is regulated by various stimuli; however,whether these stimuli regulate the same or different precursor populations remains unknown. Here,we developed a novel cell-sorting protocol that allows the purification to homogeneity of neurosphere-forming neural precursors from the adult mouse hippocampus and examined the responsiveness of individual precursors to various stimuli using a clonal assay. We show that within the Hes5-GFP(+)/Nestin-GFP(+)/EGFR(+) cell population,which comprises the majority of neurosphere-forming precursors,there are two distinct subpopulations of quiescent precursor cells,one directly activated by high-KCl depolarization,and the other activated by norepinephrine (NE). We then demonstrate that these two populations are differentially distributed along the septotemporal axis of the hippocampus,and show that the NE-responsive precursors are selectively regulated by GABA,whereas the KCl-responsive precursors are selectively modulated by corticosterone. Finally,based on RNAseq analysis by deep sequencing,we show that the progeny generated by activating NE-responsive versus KCl-responsive quiescent precursors are molecularly different. These results demonstrate that the adult hippocampus contains phenotypically similar but stimulus-specific populations of quiescent precursors,which may give rise to neural progeny with different functional capacity. View Publication

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung,liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells,hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2,SOX17,HNF1B,members of the GATA family,and the surface receptor CXCR4. However,differentiation protocols are rarely 100% efficient. Here,we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations. View Publication

The transcription factor Runx1/AML1 is an important regulator of hematopoiesis and is critically required for the generation of the first definitive hematopoietic stem cells (HSCs) in the major vasculature of the mouse embryo. As a pivotal factor in HSC ontogeny,its transcriptional regulation is of high interest but is largely undefined. In this study,we used a combination of comparative genomics and chromatin analysis to identify a highly conserved 531-bp enhancer located at position + 23.5 in the first intron of the 224-kb mouse Runx1 gene. We show that this enhancer contributes to the early hematopoietic expression of Runx1. Transcription factor binding in vivo and analysis of the mutated enhancer in transient transgenic mouse embryos implicate Gata2 and Ets proteins as critical factors for its function. We also show that the SCL/Lmo2/Ldb-1 complex is recruited to the enhancer in vivo. Importantly,transplantation experiments demonstrate that the intronic Runx1 enhancer targets all definitive HSCs in the mouse embryo,suggesting that it functions as a crucial cis-regulatory element that integrates the Gata,Ets,and SCL transcriptional networks to initiate HSC generation. View Publication

OBJECTIVE: In murine hematopoietic tissue,direct repopulation experiments have demonstrated that the side population (SP) represents a remarkable enrichment of hematopoietic stem cells. Human SP has been phenotyped as negative for lineage antigens as well as CD34. However,in the 9 years since the original publication,no long-term hematopoietic reconstitution has been reported for the adult human SP/CD34(-) subset. Elevated levels of aldehyde dehydrogenase (ALDH) have been demonstrated in murine and human progenitor cells when compared to other hematopoietic cells. METHODS: Here,we report the phenotype of human cord blood SP cells. We established the technique of simultaneous phenotyping,Hoechst exclusion,and ALDH labeling on murine tissues. We then performed the simultaneous analysis of phenotype,SP,and ALDH activity on human cord blood and bone marrow cells. Finally,we analyzed the phenotype and functional potential of human cord blood ALDH(+) cells to determine whether Lin(-)/CD34(-) cells are identified via this technique. RESULTS: We demonstrate that human Lin(-)/CD34(-)/ALDH(+) cells are capable of long-term repopulation. Although the SP technique identifies cells that overlap with the ALDH(+) cell population,this is restricted to the CD34(+) cell subset. CONCLUSION: Hoechst exclusion ability does not seem to be the method of choice for the isolation of human hematopoietic stem cells. View Publication

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging,however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS),DNA damage,senescence,and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC,as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically,CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress,as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC),improved their niche supporting activity,and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production,driving an HSC aging phenotype. Thus,modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. View Publication

Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation,sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci,cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells,with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors. View Publication