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

BackgroundSickle cell disease (SCD) and β-thalassemia patients with elevated gamma globin (HBG1/G2) levels exhibit mild or no symptoms. To recapitulate this natural phenomenon,the most coveted gene therapy approach is to edit the regulatory sequences of HBG1/G2 to reactivate them. By editing more than one regulatory sequence in the HBG promoter,the production of fetal hemoglobin (HbF) can be significantly increased. However,achieving this goal requires precise nucleotide conversions in hematopoietic stem and progenitor cells (HSPCs) at therapeutic efficiency,which remains a challenge.MethodsWe employed Cas9 RNP-ssODN-mediated homology-directed repair (HDR) gene editing to mimic two naturally occurring HBG promoter point mutations; -175T > C,associated with high HbF levels,and −158 C > T,a common polymorphism in the Indian population that induces HbF under erythropoietic stress,in HSPCs.ResultsAsymmetric,nontarget ssODN induced high rates of complete HDR conversions,with at least 15% of HSPCs exhibiting both the −175T > C and −158 C > T mutations. Optimized conditions and treatment with the small molecule AZD-7648 increased this rate,with up to 57% of long-term engrafting human HSPCs in NBSGW mice containing at least one beneficial mutation. Functionally,in vivo erythroblasts exhibited high levels of HbF,which was sufficient to reverse the cellular phenotype of β-thalassemia. Further support through bone marrow MSC co-culture boosted complete HDR conversion rates to exceed 80%,with minimal InDels,improved cell viability,and induced fetal hemoglobin levels similar to those of Cas9 RNP-mediated indels at BCL11A enhancer and HBG promoter.ConclusionsCas9 RNP-ssODN-based nucleotide conversion at the HBG promoter offers a promising gene therapy approach to ameliorate the phenotypes of β-thalassemia and SCD. The developed approach can simplify and broaden applications that require the cointroduction of multiple nucleotide modifications in HSPCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-04117-0. View Publication

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties,Sca-1 expression (Sca-1+),lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-),and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in textgreater or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients,respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor,interleukin-6 (IL-6),and erythropoietin (with or without IL-3),a large increase in total cell number,including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly,this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo,as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture,providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype. View Publication

Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. In this study,we investigated the role of HES1,a member of HES family,in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry,histopathology analysis,and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers,decreases hematopoietic stem progenitor cell (HSPC) pool,results in defective multi-lineage differentiation. Functionally,fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1 fl/fl Flt3Cre embryos are resulted from decreased proliferation and elevated apoptosis,associated with de-repressed HES1 targets,p27 and PTEN in Hes1 -KO fetal HSPCs. Finally,pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. Together,our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis,and provide new insight into the differences between fetal and adult HSC maintenance. The online version contains supplementary material available at 10.1186/s13287-024-03836-8. 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

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However,the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study,we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow,in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells,NK progenitors,and NKG2A-negative NK cells),and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore,we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally,we show that acetylated histones are associated with the CpG-rich region in NKG2A positive,but not negative,cell lines,and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription,but cotreatment with both drugs resulted in a significantly greater induction,suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells. View Publication

Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins,in particular albumin,which is prone to quality variations and presents a substantial cost factor,hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations,we have developed chemically defined,entirely protein-free media based on RPMI,supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). Both media compositions enable the efficient,directed differentiation of embryonic and induced hPSCs,matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture,producing > 1.3 × 10 9 cardiomyocytes in a single process run. Transcriptome analysis,flow cytometry,electrophysiology,and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. This study promotes the robustness and upscaling of the cardiomyogenic differentiation process,substantially reduces media costs,and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration. The online version contains supplementary material available at 10.1186/s13287-024-03826-w. View Publication

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However,scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard,suspension cultures are a viable alternative,because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However,the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here,we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production. ?? 2014 The Authors. View Publication

Hematopoietic stem cells (HSCs) have the capacity to self-renew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols,the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines. View Publication

BACKGROUND: ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD: We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br),and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations),to develop into several cell lineages in culture. RESULTS: The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD,n=30) nucleated cells and was enriched in cells expressing CD34,CD117,CD105,CD127,CD133 and CD166,and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells,and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes,osteoblasts and chondrocytes. DISCUSSION: Hematopoietic,endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications. View Publication