Oncostatin M maintains the hematopoietic microenvironment in the bone marrow by modulating adipogenesis and osteogenesis

Abstract

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.

Figure 1. Impaired BM hematopoiesis in OSM KO mice.

(A) Hematocrit values of WT (dashed line) and OSM KO mice (solid line) after Spx treatment. Hematocrit values in Spx-treated OSM KO mice showed no sign of recovery from anemic symptoms. Data are presented as means ± S.E.M. (n = 4). (B) Plasma concentrations of EPO in WT (dashed line) and OSM KO mice (solid line) by ELISA. Data are presented as means ± S.E.M. (n = 4). (C) Gene expression analysis of IL-6 family cytokines (OSM, IL-6, LIF, and CNTF) in the BM of WT mice. The expression level was normalized against that of β-actin. Data are presented as means ± S.D. (n = 4). (D) Real-time RT-PCR analysis of OSM mRNA expression in CD45⁺ Ter119⁺ cells (blood cells) and CD45^-Ter119^- cells (the other cells) of the BM. *P<0.05, **P<0.01, ***P<0.001. N.D., not detected.

Figure 2. Adipogenic BM of OSM KO mice by aging and injury.

(A) Oil Red O staining of femurs of young (10-week-old) and aged (32-week-old) WT and OSM KO mice (14-µm frozen sections). (B) Expression of adipogenesis-related genes by real-time RT-PCR in the BM of 10- and 32-weeks-old WT and OSM KO mice (n = 5). (C) The experimental design of chemotherapy-induced myeloablation. WT and OSM KO mice were splenectomized and then treated with an intraperitoneal injection of 20 mg/kg busulfan three times. After the final injection the hematocrit value was monitored weekly for 3 weeks. (D) Oil Red O staining of femur sections from WT and OSM KO mice 7 days after myeloablation. (E) Real-time RT-PCR analyses of the genes related to adipogenesis. The expression levels of adipsin and perilipin mRNA are shown (n = 4). (F) Hematocrit values after busulfan treatment of WT (open circle) and OSM KO mice (closed circle) (WT: 0–7 days, n = 8; 14–21 days, n = 3; OSM KO: 0–7 days, n = 5; 14–21 wks, n = 3). Data are shown as means ± S.E.M. *P<0.05, **P<0.01. Scale bars indicate 100 µm.

Figure 3. Multiple roles of OSM in differentiation of PαS cells in vitro.

(A) Evaluation of lipid accumulation in the cells by Oil Red O staining. Adipocytic-differentiated cells are indicated by arrowheads. (B) Real-time RT-PCR analysis of genes related to adipocytic differentiation. The expression levels of adipsin, and perilipin mRNA are shown (n = 4). (C) The expression profiles of osteogenesis-related genes after induction of osteogenic differentiation. The expression levels of Alpl, Spp1 and Bglap2 were measured by real-time RT-PCR after 5, 12, and 20 days of osteogenic induction in the presence (closed circle) or absence (open circle) of 10 ng/mL OSM. (D) Alizarin Red S staining after 15 days and 24 days of induction. (E) Real-time RT-PCR analysis of Alpl, Spp1 and Bglap2 after 7 days of culture with Dex (n = 3). Data are presented as means ± S.D. *P<0.05, **P<0.01, ***P<0.001. Scale bars indicate 100 µm.

Figure 4. OSM enhances the capacity of PαS-derived osteoblastic cells to support hematopoisis in vitro.

(A) The experimental schedule for the co-culture of LSK cells and the feeder layer. After the osteoblastic differentiation of PαS cells with or without OSM, LSK cells were co-cultured under 4% O₂ conditions (balanced by N₂). After 7 days of co-culture without OSM, cells were harvested and reanalyzed by FACS. (B) The morphology of LSK cells cultured on Oc-feeder and OSM-Oc-feeder. “Cobblestone”-like clusters (arrows) were observed in the OSM-Oc-feeder. Scale bars indicate 100 µm. (C) FACS analysis of harvested cells after 7 days of co-culture. Representative images and the percentage of the LSK cells in lineage negative CD45+ cells are shown. (D) The total numbers of expanded cells after 7 days of co-culture (n = 5). (E, F) The percentage of the LSK cells in total CD45 positive cells (E) and fold increase of LSK cells relative to input cells (F) after 7 days of co-culture are shown (n = 5). (G) Expression analysis of TPO in the Oc-feeder and OSM-Oc-feeder by real-time RT-PCR (n = 3). Data are presented as means ± S.D. **P<0.01, ***P<0.001.

Figure 5. OSM suppresses fatty marrow and enhances the recovery of BM microenvironment after irradiation in vivo.

(A) The experimental schedule for irradiation and OSM administrations. BM cells were transplanted into lethally irradiated WT mice by tail vein injection. A dose of 600 ng OSM per mouse was injected intraperitoneally twice a day for 7 days. (B) Oil red O staining of femur sections from PBS-treated mice (Vehicle) and OSM-treated mice. Arrow shows the open area occupied by erythrocytes. (C) Real-time RT-PCR analysis of genes related to adipocytic differentiation. The expression levels of adipsin and perilipin mRNA are shown (n = 4-5 per group). (D) Expression analysis of TPO in the BM by real-time RT-PCR (Vehicle, n = 4; OSM-treated mice, n = 5). (E) The total number of BM cells per a femur after 14 days of BMT. (F) FACS analysis of BM cells in vehicle-treated and OSM-treated mice. Representative images and the percentage of LSK cells in BM cells are shown. (G) The percentage of LSK cell in BM cells. (H) The LSK number in the BM per a femur. (Vehicle, n = 6; OSM-treated mice, n = 7). Data are shown as means ± S.E.M. *P<0.05, **P<0.01, ***P<0.001.Scale bars represent 100 µm.

Figure 6. OSM enhances the recovery of BM hematopoiesis after irradiation in vivo.

(A) The experimental schedule for splenectomy, irradiation and OSM administrations. WT mice were irradiated at lethal dose after 14 days of splenectomy and then a dose of 600 ng OSM per mouse was injected intraperitoneally twice a day for 7 days. Blood samples were harvested from tail vein and analyzed by automated counter every 7 days. (B-D) Hematologic analyses of peripheral blood after BMT. While blood cell count (WBC) (B) platelet cell count (PLT) (C) were measured by an automated counter. (D) Red blood cell count (RBC), mean corpuscular volume (MCV), hemoglobin content (HGB), and hematocrit values (HCT) are shown. (E) Model of multiple regulatory roles of OSM in the BM stromal cell differentiation and hematopoietic microenvironment. Data are shown as means ± S.E.M. (n = 5 per group). *P<0.05, **P<0.01, ***P<0.001.