Cytochrome P4501B1 in bone marrow is co-expressed with key markers of mesenchymal stem cells. BMS2 cell line models PAH disruption of bone marrow niche development functions.
ABSTRACT: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6?h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b?>?Tnfa> Ifng) within 6?h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined.
Project description:Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) niche and serve as a reservoir for mature blood cells throughout life. Aging in the BM is characterized by low-grade chronic inflammation that could contribute to the reduced functionality of aged HSPC. Mesenchymal stromal cells (MSC) in the BM support HSPC self-renewal. However, changes in MSC function with age and the crosstalk between MSC and HSPC remain understudied. Here, we conducted an extensive characterization of senescence features in BM-derived MSC from young and aged healthy donors. Aged MSC displayed an enlarged senescent-like morphology, a delayed clonogenic potential and reduced proliferation ability when compared to younger counterparts. Of note, the observed proliferation delay was associated with increased levels of SA-?-galactosidase (SA-?-Gal) and lipofuscin in aged MSC at early passages and a modest but consistent accumulation of physical DNA damage and DNA damage response (DDR) activation. Consistent with the establishment of a senescence-like state in aged MSC, we detected an increase in pro-inflammatory senescence-associated secretory phenotype (SASP) factors, both at the transcript and protein levels. Conversely, the immunomodulatory properties of aged MSC were significantly reduced. Importantly, exposure of young HSPC to factors secreted by aged MSC induced pro-inflammatory genes in HSPC and impaired HSPC clonogenic potential in a SASP-dependent manner. Altogether, our results reveal that BM-derived MSC from aged healthy donors display features of senescence and that, during aging, MSC-associated secretomes contribute to activate an inflammatory transcriptional program in HSPC that may ultimately impair their functionality.
Project description:Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1? as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1? alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.
Project description:Bone marrow (BM) hematopoietic stem cells differentiate to common lymphoid progenitors (CLP) that emigrate to the thymus to form T cells or differentiate into immature B cells that then migrate to the spleen for maturation. Rapid in vivo suppression of BM progenitor cells by a single oral or intraperitoneal dose of 7,12-dimethylbenz(a)anthracene (DMBA) subsequently decreased mature lymphoid populations in BM, spleen, and thymus. These suppressions depended on BM CYP1B1, but not on aryl hydrocarbon receptor (AhR) activity. Suppression of pre-B colony formation at 6 h, correlated with subsequent decreases in mature BM, spleen, and thymus populations (48-168 h). Thymus T-cell ratios were unaffected, suggesting low local toxicity. DMBA treatment suppressed progenitor cells 24-h post treatment in wild type (WT), AhRb mice, but not in Cyp1b1-ko mice. The stem cell populations were sustained. Benzo(a)pyrene (BP) mediated a similar progenitor suppression up to 6 h, but reversal rapidly ensued. This recovery was absent in mice with a polycyclic aromatic hydrocarbon (PAH)-resistant, AhRd genotype. This AhR-dependent progenitor recovery with BP induction accounts for the absence of suppression of B220+ BM and spleen populations at 48-168 h. However, DMBA and BP produced similar profiles for thymus cell suppression, independent of AhR genotype. Thus, lymphoid progenitors may exit the BM to the thymus prior to the BP reversal. This progenitor recovery is associated with elevated chemokines and cytokines that depend on AhR-mediated induction of CYP1A1. This response increased constitutively in Cyp1b1-ko BM, demonstrating that CYP1B1 metabolizes local stimulants that impact a basal progenitor protection process.
Project description:Mesenchymal stromal cells (MSCs) are key elements in the bone marrow (BM) niche where they interact with hematopoietic stem progenitor cells (HSPCs) by offering physical support and secreting soluble factors, which control HSPC maintenance and fate. Although necessary for their maintenance, MSCs are a rare population in the BM, they are plastic adherent and can be ex vivo expanded to reach numbers adequate for clinical use. In light of HSPC supportive properties, MSCs have been employed in phase I/II clinical trials of hematopoietic stem cell transplantation (HSCT) to facilitate engraftment of hematopoietic stem cells (HSCs). Moreover, they have been utilized to expand ex vivo HSCs before clinical use. The available clinical evidence from these trials indicate that MSC administration is safe, as no acute and long-term adverse events have been registered in treated patients, and may be efficacious in promoting hematopoietic engraftment after HSCT. In this review, we critically discuss the role of MSCs as component of the BM niche, as recent advances in defining different mesenchymal populations in the BM have considerably increased our understanding of this complex environment. Moreover, we will revise published literature on the use of MSCs to support HSC engraftment and expansion, as well as consider potential new MSC application in the clinical context of ex vivo gene therapy with autologous HSC.
Project description:Sirtuin 1 (SIRT1) is a histone deacetylase implicated in stem cell homeostasis. Conditional Sirt1 deletion in the hematopoietic stem and progenitor system promotes hematopoietic stem and progenitor cell (HSPC) expansion under stress conditions. In addition, SIRT1 activators modulate the capacity and HSPC numbers in the bone marrow (BM). To investigate the role of SIRT1 in the BM niche, a conditional Sirt1 deletion in the BM niche was generated in a mouse model for the present study. Multicolor flow cytometric analyses were performed to determine HSC cell populations. Using 5-fluorouracil-induced proliferative stress, a survival curve was produced. In the present study, Sirt1 deletion in the BM niche demonstrated that the production of mature blood cells, lineage distribution within hematopoietic organs and frequencies of HSPC populations were comparable to those of controls. Additionally, Sirt1 deletion in the BM niche did not perturb HSC maturation under stress induced by transplantation. Therefore, these observations suggest that SIRT1 serves a dispensable role in HSC maturation in the BM niche.
Project description:An understanding of the cell interactions occurring in the leukemic microenvironment and their functional consequences for the different cell players has therapeutic relevance. By co-culturing mesenchymal stem cells (MSC) with the REH acute lymphocytic leukemia (ALL) cell line, we have established an in vitro leukemic niche for the functional evaluation of hematopoietic stem/progenitor cells (HSPC, CD34+ cells). We showed that the normal homeostatic control exerted by the MSC over the HSPC is considerably lost in this leukemic microenvironment: HSPC increased their proliferation rate and adhesion to MSC. The adhesion molecules CD54 and CD44 were consequently upregulated in HSPC from the leukemic niche. Consequently, with this adhesive phenotype, HSPC showed less Stromal derived factor-1 (SDF-1)-directed migration. Interestingly, multipotency was severely affected with an important reduction in the absolute count and the percentage of primitive progenitor colonies. It was possible to simulate most of these HSPC alterations by incubation of MSC with a REH-conditioned medium, suggesting that REH soluble factors and their effect on MSC are important for the observed changes. Of note, these HSPC alterations were reproduced when primary leukemic cells from an ALL type B (ALL-B) patient were used to set up the leukemic niche. These results suggest that a general response is induced in the leukemic niche to the detriment of HSPC function and in favor of leukemic cell support. This in vitro leukemic niche could be a valuable tool for the understanding of the molecular events responsible for HSPC functional failure and a useful scenario for therapeutic evaluation.
Project description:Hematopoietic stem and progenitor cell (HSPC) homeostasis declines with age, leading to impaired hematopoiesis. Mesenchymal stromal cells (MSC) are critical components of the bone marrow niche and key regulators of the balance between HSPC proliferation and quiescence. Accrual of DNA damage, a hallmark of cellular aging, occurs in aged MSC. Whether MSC aging alters the bone marrow niche triggering HSPC dysfunction is unknown. Using a human MSC-HSPC co-culture system, we demonstrated that DNA damaged MSC have impaired capacity to maintain CD34+CD38- HSPC quiescence. Furthermore, human MSC from adult donors display some hallmarks of cellular senescence and have a decreased capacity to maintain HSPC quiescence and the most primitive CD34+CD38- subset compared to MSC from pediatric donors. IL-6 neutralization restores the MSC-HPSC crosstalk in senescent and adult MSC-HSPC co-cultures highlighting the relevance of the local microenvironment in maintaining HSPC homeostasis. These results provide new evidence implicating components of the MSC secretome in HSPC aging.
Project description:The bone marrow (BM) is an essential organ for hematopoiesis in adult, in which proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) is orchestrated by various stromal cells. Alterations of BM hematopoietic environment lead to various hematopoietic disorders as exemplified by the linking of fatty marrow with increased adipogenesis to anemia or pancytopenia. Therefore, the composition of mesenchymal stromal cell (MSC)-derived cells in the BM could be crucial for proper hematopoiesis, but the mechanisms underlying the MSC differentiation for hematopoiesis remain poorly understood. In this study, we show that Oncostatin M (OSM) knock out mice exhibited pancytopenia advancing fatty marrow with age. OSM strongly inhibited adipogenesis from BM MSC in vitro, whereas it enhanced their osteogenesis but suppressed the terminal differentiation. Intriguingly, OSM allowed the MSC-derived cells to support the ex vivo expansion of HSPC effectively as feeder cells. Furthermore, the administration of OSM in lethally irradiated wild-type mice blocked fatty marrow and enhanced the recovery of HSPC number in the BM and peripheral blood cells after engraftment of HSPC. Collectively, OSM plays multiple critical roles in the maintenance and development of the hematopoietic microenvironment in the BM at a steady state as well as after injury.
Project description:The interactions of hematopoietic stem and progenitor cells (HSPCs) with extracellular matrix (ECM) components and cells from the bone marrow (BM) microenvironment control their homeostasis. Regenerative BM conditions can induce expression of the ECM protein transforming growth factor beta-induced gene H3 (TGFBI or BIGH3) in murine HSPCs. In this study, we examined how increased or reduced TGFBI expression in human HSPCs and BM mesenchymal stromal cells (MSCs) affects HSPC maintenance, differentiation, and migration. HSPCs that overexpressed TGFBI showed accelerated megakaryopoiesis, whereas granulocyte differentiation and proliferation of granulocyte, erythrocyte, and monocyte cultures were reduced. In addition, both upregulation and downregulation of TGFBI expression impaired HSPC colony-forming capacity of HSPCs. Interestingly, the colony-forming capacity of HSPCs with reduced TGFBI levels was increased after long-term co-culture with MSCs, as measured by long-term culture-colony forming cell (LTC-CFC) formation. Moreover, TGFBI downregulation in HSPCs resulted in increased cobblestone area-forming cell (CAFC) frequency, a measure for hematopoietic stem cell (HSC) capacity. Concordantly, TGFBI upregulation in HSPCs resulted in a decrease of CAFC and LTC-CFC frequency. These results indicate that reduced TGFBI levels in HSPCs enhanced HSC maintenance, but only in the presence of MSCs. In addition, reduced levels of TGFBI in MSCs affected MSC/HSPC interaction, as observed by an increased migration of HSPCs under the stromal layer. In conclusion, tight regulation of TGFBI expression in the BM niche is essential for balanced HSPC proliferation and differentiation.
Project description:Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) stem cell niche, which provides a vital source of HSC regulatory signals. Radiation and chemotherapy disrupt the HSC niche, including its sinusoidal vessels and perivascular cells, contributing to delayed hematopoietic recovery. Thus, identification of factors that can protect the HSC niche during an injury could offer a significant therapeutic opportunity to improve hematopoietic regeneration. In this study, we identified a critical function for vascular endothelial growth factor-C (VEGF-C), that of maintaining the integrity of the BM perivascular niche and improving BM niche recovery after irradiation-induced injury. Both global and conditional deletion of Vegfc in endothelial or leptin receptor-positive (LepR+) cells led to a disruption of the BM perivascular niche. Furthermore, deletion of Vegfc from the microenvironment delayed hematopoietic recovery after transplantation by decreasing endothelial proliferation and LepR+ cell regeneration. Exogenous administration of VEGF-C via an adenoassociated viral vector improved hematopoietic recovery after irradiation by accelerating endothelial and LepR+ cell regeneration and by increasing the expression of hematopoietic regenerative factors. Our results suggest that preservation of the integrity of the perivascular niche via VEGF-C signaling could be exploited therapeutically to enhance hematopoietic regeneration.