Murine Sca1(+)Lin(-) bone marrow contains an endodermal precursor population that differentiates into hepatocytes.
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ABSTRACT:
SUBMITTER: Oh K
PROVIDER: S-EPMC4673473 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:Murine very small embryonic-like (VSEL) cells, defined by the Lin(-)Sca-1(+)CD45(-) phenotype and small size, were described as pluripotent cells and proposed to be the most primitive hematopoietic precursors in adult bone marrow. Although their isolation and potential application rely entirely on flow cytometry, the immunophenotype of VSELs has not been extensively characterized. Our aim was to analyze the possible heterogeneity of Lin(-)Sca(+)CD45(-) population and investigate the extent to which VSELs characteristics may overlap with that of hematopoietic stem cells (HSCs) or endothelial progenitor cells (EPCs). The study evidenced that murine Lin(-)Sca-1(+)CD45(-) population was heterogeneous in terms of c-Kit and KDR expression. Accordingly, the c-Kit(+)KDR(-), c-Kit(-)KDR(+), and c-Kit(-)KDR(-) subpopulations could be distinguished, while c-Kit(+)KDR(+) events were very rare. The c-Kit(+)KDR(-) subset contained almost solely small cells, meeting the size criterion of VSELs, in contrast to relatively bigger c-Kit(-)KDR(+) cells. The c-Kit(-)KDR(-)FSC(low) subset was highly enriched in Annexin V-positive, apoptotic cells, hence omitted from further analysis. Importantly, using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin(-)Sca-1(+)CD45(-)FSC(low) population, even by single-cell qRT-PCR. We also found that the Lin(-)Sca-1(+)CD45(-)c-Kit(+) subset did not exhibit hematopoietic potential in a single cell-derived colony in vitro assay, although it comprised the Sca-1(+)c-Kit(+)Lin(-) (SKL) CD34(-)CD45(-)CD105(+) cells, expressing particular HSC markers. Co-culture of Lin(-)Sca-1(+)CD45(-)FSC(low) with OP9 cells did not induce hematopoietic potential. Further investigation revealed that SKL CD45(-)CD105(+) subset consisted of early apoptotic cells with fragmented chromatin, and could be contaminated with nuclei expelled from erythroblasts. Concluding, murine bone marrow Lin(-)Sca-1(+)CD45(-)FSC(low) cells are heterogeneous population, which do not express the pluripotency marker Oct-4A. Despite expression of some hematopoietic markers by a Lin(-)Sca-1(+)CD45(-)c-Kit(+)KDR(-) subset of VSELs, they do not display hematopoietic potential in a clonogenic assay and are enriched in early apoptotic cells.
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Project description:Bone-marrow transplantation (BMT) can repopulate the liver through BM-derived hepatocyte (BMDH) generation, although the underlying mechanism remains unclear. Using fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mice as a liver-failure model, we confirmed that BMDHs were generated by fusion of BM-derived CD11b(+)F4/80(+)myelomonocytes with resident Fah(-/-) hepatocytes. Hepatic NK cells became activated during BMDH generation and were the major IFN-γ producers. Indeed, both NK cells and IFN-γ were required for BMDH generation since WT, but not NK-, IFN-γ-, or IFN-γR1-deficient BM transplantation successfully generated BMDHs and rescued survival in Fah(-/-) hosts. BM-derived myelomonocytes were determined to be the IFN-γ-responding cells. The IFN-γ-IFN-γR interaction contributed to the myelomonocyte-hepatocyte fusion process, as most of the CD11b(+) BMDHs in mixed BM chimeric Fah(-/-) hosts transplanted with a 1:1 ratio of CD45.1(+) WT and CD45.2(+) Ifngr1(-/-) BM cells were of CD45.1(+) WT origin. Confirming these findings in vitro, IFN-γ dose-dependently promoted the fusion of GFP(+) myelomonocytes with Fah(-/-) hepatocytes due to a direct effect on myelomonocytes; similar results were observed using activated NK cells. In conclusion, BMDH generation requires NK cells to facilitate myelomonocyte-hepatocyte fusion in an IFN-γ-dependent manner, providing new insights for treating severe liver failure.
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Project description:IntroductionEarly recovery of donor-derived invariant natural killer T (iNKT) cells are associated with reduced risk of graft-versus-host disease (GvHD) and overall survival. Patients with severe GvHD, however, had much slower iNKT cell reconstitution relative to conventional T cells.MethodsTo characterize the delay of iNKT cell reconstitution and explore its possible causes, we used a haploidentical bone marrow transplantation (haplo-BMT) mouse model with GvHD. We found the delayed recovery of thymic and peripheral iNKT cell numbers with markedly decreased thymic NKT1 subset in GvHD mice. The defective generation of thymic iNKT precursors with egress capability contributed to the reduced peripheral iNKT cells in GvHD mice. We further identified intermediate NK1.1- NKT1 precursor subpopulations under steady-state conditions and found that the differentiation of these subpopulations was impaired in the thymi of GvHD mice. Detailed characterization of iNKT precursors and thymic microenvironment showed a close association of elevated TCR/co-stimulatory signaling provided by double positive thymocytes and macrophages with defective down-regulation of proliferation, metabolism, and NKT2 signature in iNKT precursor cells. Correspondingly, NKT2 but not NKT1 differentiation was favored in GvHD mice.DiscussionThese data underline the important roles of TCR and co-stimulatory signaling in the differentiation of thymic iNKT subsets under transplantation conditions.
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Project description:The aim of this study was to characterize in vivo miRNA expression in normal myeloid development of the neutrophil granulocyte (granulopoiesis) to gain insight into miRNA control of these processes. Cell populations highly enriched in precursors from successive stages of granulopoiesis and mature neutrophils were isolated from bone marrow (BM) and peripheral blood (PB) samples, respectively, from 4 healthy human donors. Total RNA was extracted from each cell population and subjected to miRNA gene expression profiling using miRCURYTM LNA microRNA Arrays
2011-10-22 | GSE33140 | GEO
Project description:The aim of this study was to characterize in vivo miRNA expression in normal myeloid development of the neutrophil granulocyte (granulopoiesis) to gain insight into miRNA control of these processes. Cell populations highly enriched in precursors from successive stages of granulopoiesis and mature neutrophils were isolated from bone marrow (BM) and peripheral blood (PB) samples, respectively, from 4 healthy human donors. Total RNA was extracted from each cell population and subjected to miRNA gene expression profiling using miRCURYTM LNA microRNA Arrays Total RNA from cell populations highly enriched in precursors from three succesive stages of neutrophil granulopoiesis and mature neutrophils isolated from bone marrow and peripheral blood, respectively, from four healthy donors (SKP, AJ, AO, JO). The three cell populations from bone marrow where extracted and named B3, B2, and B1, corresponding to immature (myeloblasts [MBs] and promyelocytes [PMs]); intermediate mature (myelocytes [MCs] and metamyelocytes [MMs]); and mature neutrophil cells (band cells [BCs] and segmented neutrophil cells [SCs]), respectively, as described in Bjerregaard MD, Jurlander J, Klausen P, Borregaard N, Cowland JB: The in vivo profile of transcription factors during neutrophil differentiation in human bone marrow. Blood 2003, 101: 4322-4332. In total, 16 samples were compared (4 cell maturation stages from 4 donors)
2011-10-21 | E-GEOD-33140 | biostudies-arrayexpress
Project description:We previously reported that diabetes in mice is associated with the appearance of proinsulin-producing (Proins-P) cells in the liver. It was unclear, however, whether these Proins-P bone marrow-derived cells (BMDC) merely transit through the liver or undergo fusion with hepatocytes, normally an extremely rare event. In this study, we found that, in diabetes, BMDC in the liver produce not only Proins but also TNF-alpha, suggesting that diabetes reprograms gene expression in BMDC, turning on "inappropriate" genes. Bone marrow transplantation using genetically marked donor and recipient mice showed that fusion occurs between Proins-P BMDC and hepatocytes. Cell fusion is further supported by the presence of the Y chromosome in Proins-P cells in female mice that received male bone marrow transplantation cells. Morphologically, Proins-P fusion cells are albumin-producing hepatocytes that constitute approximately 2.5% of the liver section area 5 months after diabetes induction. An extensive search failed to reveal any fusion cells in nondiabetic mice. Thus, diabetes causes fusion between Proins-P BMDC and hepatocytes in vivo, an observation that has implications for the pathophysiology of diabetes as well as the fundamental biology of heterotypic cell fusion.
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Project description:Eosinophils are produced in the bone marrow from CD34+ eosinophil lineage-committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage-committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage-committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineage-committed progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineage-committed progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5.
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Project description:CD4(+) T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4(+) T cell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9(+) multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4(+) T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4(+) cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, and total CD4(+) T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4(+) T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9(+) MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9(+) MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4(+) T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection.
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Project description:Dendritic cells (DCs) play a crucial role in bridging innate and adaptive immunity by activating naïve T cells. The role of pattern recognition receptors like Toll-Like Receptors and Nod-Like Receptors expressed on DCs is well-defined in the recognition of the pathogens. However, nothing is precisely studied regarding the impact of NOD-2 signaling during the differentiation of DCs. Consequently, we explored the role of NOD-2 signaling in the differentiation of DCs and therefore their capability to activate innate and adaptive immunity. Intriguingly, we observed that NOD-2 stimulated DCs (nDCs) acquired highly activated and matured phenotype and exhibited substantially greater bactericidal activity by robust production of nitric oxide. The mechanism involved in improving the functionality of nDCs was dependent on IFN-αβ signaling, leading to the activation of STAT pathways. Furthermore, we also observed that STAT-1 and STAT-4 dependent maturation and activation of DCs was under the feedback mechanism of SOCS-1 and SOCS-3 proteins. nDCs acquired enhanced potential to activate chiefly Th1 and Th17 immunity. Taken together, these results suggest that nDCs can be exploited as an immunotherapeutic agent in bolstering host immunity and imparting protection against the pathogens.
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