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

Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here,we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs,which are the most potent subsets,it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol,please refer to Liang et al. (2020) and Qiu et al. (2021). View Publication

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH,however,lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore,other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs),characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming,and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation,the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients. View Publication

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58{\%} Venus(+) HSPCs with 6-16{\%} human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD),caused by mutations in the gp91phox subunit of the NADPH oxidase,TI of a gp91phox transgene into AAVS1 resulted in ∼15{\%} gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs,4-11{\%} of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases. View Publication

Both extrinsic and intrinsic mechanisms tightly govern hematopoietic stem cell (HSC) decisions of self-renewal and differentiation. However,transcription factors that can selectively regulate HSC self-renewal division after stress remain to be identified. Slug is an evolutionarily conserved zinc-finger transcription factor that is highly expressed in primitive hematopoietic cells and is critical for the radioprotection of these key cells. We studied the effect of Slug in the regulation of HSCs in Slug-deficient mice under normal and stress conditions using serial functional assays. Here,we show that Slug deficiency does not disturb hematopoiesis or alter HSC homeostasis and differentiation in bone marrow but increases the numbers of primitive hematopoietic cells in the extramedullary spleen site. Deletion of Slug enhances HSC repopulating potential but not its homing and differentiation ability. Furthermore,Slug deficiency increases HSC proliferation and repopulating potential in vivo after myelosuppression and accelerates HSC expansion during in vitro culture. Therefore,we propose that Slug is essential for controlling the transition of HSCs from relative quiescence under steady-state condition to rapid proliferation under stress conditions. Our data suggest that inhibition of Slug in HSCs may present a novel strategy for accelerating hematopoietic recovery,thus providing therapeutic benefits for patients after clinical myelosuppressive treatment. View Publication

Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells,its physiological function(s) remain unknown,and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations,followed by competitive bone marrow reconstitution assays,shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent,the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore,PrP is a marker for HSCs and supports their self-renewal. View Publication

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel–forming cells in dogs and humans,respectively. These vasoformative sarcomas are aggressive and highly metastatic,with disorganized,irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized,and they were lined by both donor and host cells. In a series of xenografts,we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore,gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas,and possibly human angiosarcomas,maintain molecular properties that provide hematopoietic support and facilitate stromal reactions,suggesting their potential involvement in promoting the growth of hematopoietic tumors. We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation,providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression. View Publication

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC),a characteristic of polycythemia vera and essential thrombocythemia,is not standardized. In this multicentric study,we tested four semisolid,serum-free,cytokine-free media based on either methylcellulose (M1,M2) or collagen (C1,C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26),essential thrombocythemia (19),secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without,or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific,as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia,nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria,respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo,the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay,now part of the new criteria of polycythemia vera. View Publication

We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal feeders" depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay� View Publication

Monocytic lineage cells (monocytes,macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established,these methods depend on the use of xenogeneic materials and,therefore,have a relatively poor-reproducibility. Here,we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6) ± 0.3 × 10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine,disease-specific iPSC studies and drug discovery. View Publication

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as $\beta$-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex,consisting of purified Cas9 protein and guide RNA,into HSPCs. Because $\beta$-hemoglobinopathies are most prevalent in developing countries,it is desirable to have a reliable,efficient,easy-to-use and cost effective delivery method. With this goal in mind,we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF),a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale. View Publication

CD4 T cells play a critical role in promoting the development of autoimmunity in type 1 diabetes. The diabetogenic CD4 T cell clone BDC-2.5,originally isolated from a NOD mouse,has been widely used to study the contribution of autoreactive CD4 T cells and relevant Ags to autoimmune diabetes. Recent work from our laboratory has shown that the Ag for BDC-2.5 T cells is a hybrid insulin peptide (2.5HIP) consisting of an insulin C-peptide fragment fused to a peptide from chromogranin A (ChgA) and that endogenous 2.5HIP-reactive T cells are major contributors to autoimmune pathology in NOD mice. The objective of this study was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with the 2.5HIP Ag (2.5HIP-coupled PLG NPs) can tolerize BDC-2.5 T cells. Infusion of 2.5HIP-coupled PLG NPs was found to prevent diabetes in an adoptive transfer model by impairing the ability of BDC-2.5 T cells to produce proinflammatory cytokines through induction of anergy,leading to an increase in the ratio of Foxp3+ regulatory T cells to IFN-gamma+ effector T cells. To our knowledge,this work is the first to use a hybrid insulin peptide,or any neoepitope,to re-educate diabetogenic T cells and may have significant implications for the development of an Ag-specific therapy for type 1 diabetes patients. View Publication

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9(-/-)bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples,as well as in wild-type versus Hoxa9-deficient marrow,using northern analysis and qRT-PCR. Infection of wild-type BM with HOXA9 expressing or GFP(+) control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors,being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells,indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1,an important regulator of myelopoiesis,was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein,HOXA9 did not appear to modulate Pu.1 expression in murine BM cells. View Publication