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

Innate lymphoid cells 2 (ILC2) play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). An important aspect of ILC2-mediated tumorigenesis is the expansion of the resident ILC2 and simultaneous recruitment of the peripheral ILC2. Here,we describe a protocol for isolation,enrichment,and DiD labeling of ILC2 for in vivo tracking of ILC2s in the mouse. Further,we describe steps for the adoptive transfer of ILC2 to a recipient mouse model of PDAC. For complete details on the use and execution of this protocol,please refer to Alam et al. (2022). View Publication

Forced expression of MN1 in primitive mouse hematopoietic cells causes acute myeloid leukemia and impairs all-trans retinoic acid-induced granulocytic differentiation. Here,we studied the effects of MN1 on myeloid differentiation and proliferation using primary human CD34(+) hematopoietic cells,lineage-depleted mouse bone marrow cells,and bipotential (granulocytic/monocytic) human acute myeloid leukemia cell lines. We show that exogenous MN1 stimulated the growth of CD34(+) cells,which was accompanied by enhanced survival and increased cell cycle traverse in cultures supporting progenitor cell growth. Forced MN1 expression impaired both granulocytic and monocytic differentiation in vitro in primary hematopoietic cells and acute myeloid leukemia cell lines. Endogenous MN1 expression was higher in human CD34(+) cells compared with both primary and in vitro-differentiated monocytes and granulocytes. Microarray and real-time reverse-transcribed polymerase chain reaction analysis of MN1-overexpressing CD34(+) cells showed down-regulation of CEBPA and its downstream target genes. Reintroduction of conditional and constitutive CEBPA overcame the effects of MN1 on myeloid differentiation and inhibited MN1-induced proliferation in vitro. These results indicate that down-regulation of CEBPA activity contributes to MN1-modulated proliferation and impaired myeloid differentiation of hematopoietic cells. View Publication

The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs),subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB),and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However,the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study,we used the latest gene-editing tool,clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9),to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then,we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation,gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly,the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system,and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation. View Publication

The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However,investigators report studies of MSC using different methods of isolation and expansion,and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes,which hinders progress in the field. To begin to address this issue,the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First,MSC must be plastic-adherent when maintained in standard culture conditions. Second,MSC must express CD105,CD73 and CD90,and lack expression of CD45,CD34,CD14 or CD11b,CD79alpha or CD19 and HLA-DR surface molecules. Third,MSC must differentiate to osteoblasts,adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds,we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators. View Publication

Acute myelogenous leukemia 1 (AML1; runt-related transcription factor 1 [Runx1]) is a member of Runx transcription factors and is essential for definitive hematopoiesis. Although AML1 possesses several subdomains of defined biochemical functions,the physiologic relevance of each subdomain to hematopoietic development has been poorly understood. Recently,the consequence of carboxy-terminal truncation in AML1 was analyzed by the hematopoietic rescue assay of AML1-deficient mouse embryonic stem cells using the gene knock-in approach. Nonetheless,a role for specific internal domains,as well as for mutations found in a human disease,of AML1 remains to be elucidated. In this study,we established an experimental system to efficiently evaluate the hematopoietic potential of AML1 using a coculture system of the murine embryonic para-aortic splanchnopleural (P-Sp) region with a stromal cell line,OP9. In this system,the hematopoietic defect of AML1-deficient P-Sp can be rescued by expressing AML1 with retroviral infection. By analysis of AML1 mutants,we demonstrated that the hematopoietic potential of AML1 was closely related to its transcriptional activity. Furthermore,we showed that other Runx transcription factors,Runx2/AML3 or Runx3/AML2,could rescue the hematopoietic defect of AML1-deficient P-Sp. Thus,this experimental system will become a valuable tool to analyze the physiologic function and domain contribution of Runx proteins in hematopoiesis. View Publication

Autografts using untreated or in vitro manipulated bone marrow and peripheral blood stem cells represent promising approaches to the treatment of malignant diseases. In this work,the collagen gel culture technique was compared with agar and methylcellulose for its capacity to permit the growth of human granulomonocytic (day 14 CFU-GM; collagen vs agar or MTC) or erythroblastic (day 7 CFU-E and day 14 BFU-E; collagen versus methylcellulose) colonies in autologous transplantation products. Our results show that the collagen culture system always gave as many or more colonies than the other techniques. It also allowed harvesting of gels onto glass slides and subsequent May-Grünwald-Giemsa,cytochemical or immunocytochemical staining. We suggest that the collagen assay represents an interesting alternative to the widely used agar or methylcellulose systems for the culture of hematopoietic progenitors because of the equal or higher number of colonies detected,the easy phenotypical identification of colonies in stained gels,and the ability to store high-quality documentation. This technique is particularly attractive for use in the quality control of autologous bone marrow transplantation procedures. View Publication

Chimerism testing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represents a promising tool for predicting disease relapse,although its precise role in this setting remains unclear. We investigated the predictive value of T lymphocyte chimerism analysis at 90 to 120 days after allo-HSCT in 378 patients with AML/MDS who underwent busulfan/fludarabine-based myeloablative preparative regimens. Of 265 (70%) patients with available T lymphocyte chimerism data,43% of patients in first or second complete remission (CR1/CR2) at the time of transplantation had complete (100%) donor T lymphocytes at day +90 to +120 compared with 60% of patients in the non-CR1/CR2 cohort (P = .005). In CR1/CR2 patients,donor T lymphocyte chimerism ?85% at day +90 to +120 was associated with a higher frequency of 3-year disease progression (29%; 95% confidence interval [CI],18% to 46% versus 15%; 95% CI,9% to 23%; hazard ratio [HR],2.1; P = .04). However,in the more advanced,non-CR1/CR2 cohort,mixed T lymphocyte chimerism was not associated with relapse (37%; 95% CI,20% to 66% versus 34%; 95% CI,25% to 47%; HR,1.3; P = .60). These findings demonstrate that early T lymphocyte chimerism testing at day +90 to +120 is a useful approach for predicting AML/MDS disease recurrence in patients in CR1/CR2 at the time of transplantation. View Publication

Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted,but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here,we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell-autonomous manner. Unexpectedly,loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly,null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation,thereby accelerating hematopoietic regeneration. Consistent with these findings,Puma is required for radiation-induced apoptosis in HSCs and HPCs,and Puma is selectively induced by irradiation in primitive hematopoietic cells,and this induction is impaired in Puma-heterozygous cells. Together,our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy. View Publication

Ex vivo amplification of human hematopoietic stem cells (HSC) without loss of their self-renewing potential represents an important target for transplantation,gene and cellular therapies. Valproic acid is a safe and widely used neurologic agent that acts as a potent inhibitor of histone deacetylase activities. Here,we show that valproic acid addition to liquid cultures of human CD34+ cells isolated from cord blood,mobilized peripheral blood,and bone marrow strongly enhances the ex vivo expansion potential of different cytokine cocktails as shown by morphologic,cytochemical,immunophenotypical,clonogenic,and gene expression analyses. Notably,valproic acid highly preserves the CD34 positivity after 1 week (range,40-89%) or 3 weeks (range,21-52%) amplification cultures with two (Flt3L + thrombopoietin) or four cytokines (Flt3L + thrombopoietin + stem cell factor + interleukin 3). Moreover,valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4,a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133,a recognized marker gene for stem cell populations. Overall,our results relate the changes induced by valproic acid on chromatin accessibility with the enhancement of the cytokine effect on the maintenance and expansion of a primitive hematopoietic stem cell population. These findings underscore the potentiality of novel epigenetic approaches to modify HSC fate in vitro. View Publication

This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures,derived from isolated single cells,can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells,or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo,including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development. View Publication

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However,the current conventional method leads to poor recovery of hPSCs after thawing. Here,we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single-cell dissociation. After confirming hPSC survivability after freeze-thawing,we found that hPSCs that were freeze-thawed as colonies showed markedly decreased survival,whereas freeze-thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze-thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post-thawing. The improved method was also adapted to a xeno-free culture system. Moreover,the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin-521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high-quality hPSCs. View Publication

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases,both to study them and to explore therapies. However,a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study,we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV),adenoviruses and lentiviral vectors in hESC,hiPSC,and the derived cardiomyocytes. In undifferentiated cells,adenoviral and lentiviral vectors were superior,whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes,1,2,6,and 9,of which 2 and 6 were superior in their transduction efficiency. Interestingly,we observed that AAVs severely diminished the viability of undifferentiated cells,an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore,we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally,adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion,adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines,whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells. View Publication