Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment

Abstract

Osteoblasts and endothelium constitute functional niches that support haematopoietic stem cells in mammalian bone marrow. Adult bone marrow also contains adipocytes, the number of which correlates inversely with the haematopoietic activity of the marrow. Fatty infiltration of haematopoietic red marrow follows irradiation or chemotherapy and is a diagnostic feature in biopsies from patients with marrow aplasia. To explore whether adipocytes influence haematopoiesis or simply fill marrow space, we compared the haematopoietic activity of distinct regions of the mouse skeleton that differ in adiposity. Here we show, by flow cytometry, colony-forming activity and competitive repopulation assay, that haematopoietic stem cells and short-term progenitors are reduced in frequency in the adipocyte-rich vertebrae of the mouse tail relative to the adipocyte-free vertebrae of the thorax. In lipoatrophic A-ZIP/F1 'fatless' mice, which are genetically incapable of forming adipocytes, and in mice treated with the peroxisome proliferator-activated receptor-gamma inhibitor bisphenol A diglycidyl ether, which inhibits adipogenesis, marrow engraftment after irradiation is accelerated relative to wild-type or untreated mice. These data implicate adipocytes as predominantly negative regulators of the bone-marrow microenvironment, and indicate that antagonizing marrow adipogenesis may enhance haematopoietic recovery in clinical bone-marrow transplantation.

Figure 1. Hematopoietic stem cells and progenitors are reduced in number, frequency and cycling capacity in adipocyte-rich bone marrow during homeostasis

a.H&E-stain of decalcified thoracic vertebra (top) and fourth-tail-segment (bottom), 12-week-old C57BL/6J mice. b.Absolute number of hematopoietic cells (CD45+) per vertebral segment. c.Progenitor frequency within the hematopoietic compartment (CD45+). d.Competitive engraftment (250,000 CD45.1 tail or thorax BM; 250,000 CD45.2 competitor BM), e.Day 13 spleen-colony assay, and f.CFU-progenitor assay from tail and thorax BM. g.Progenitor cell cycle analysis .average % cells in S/G2/M transition ± SEM. h.100 tail and thorax BM sorted HSC (ckit+Lin-Sca1+Flk2−; >95% purity) were transplanted competitively, then engraftment in peripheral blood monitored. i.CD34 expression within the HSC fraction (KLSF, ckit+Lin-Sca1+Flk2−); % CD34low within KLSF fraction indicated.

Figure 2. The lack of bone marrow adipocytes post-irradiation in fatless mice enhances hematopoietic progenitor expansion and post-transplant recovery

a.Experimental design. Wildtype FVB or fatless FVB.A-ZIP/F 16-week-old mice (CD45.1) were lethally irradiated and transplanted with 200,000 CD45.2, MHC-compatible DBA/1 wild-type BM. Femurs were isolated on day 17–20 post-transplant and donor DBA CD45.2 wildtype BM was recovered by high purity FACS, then used for progenitor assays or competitive serial transplantation. b.Femoral H&E in the third week post-transplant. c.White blood cell (WBC) counts and d.hemoglobin levels in peripheral blood after primary transplant. BM recovered from primary transplants was assayed for e.relative frequency of progenitors by FACS (± STD) f.colony forming units assay (CFU), and g.secondary competitive transplantation into wildtype recipients.

Figure 3. Ablation of the hematopoietic compartment in fatless A-ZIP/F1 mice during BM transplantation induces osteogenesis

Analysis of mice transplanted as in figure 2. a.High-resolution microCT analysis of pre/post-transplant tibias from wildtype (top) or fatless A-ZIP/F1 mice 20 days after lethal ablation. b.Average trabecular bone density of (a) normalized to a density standard (phantom). c.Percentage BM space occupied by trabecular bone 20 days after transplantation. d.MicroPET analysis pre/post-transplant. Representative mice shown at three different timepoints (3–4 analyzed per group). Dark areas indicate NaF-18 uptake in regions of active bone deposition (red arrowheads). e.Quantification of mean NaF-18 uptake in tibiae and proximal tails pre/post-transplantation. Square and lines over micrographs a. and d. indicate quantification regions (see methods).

Figure 4. Pharmacological inhibition of adipocyte formation enhances BM engraftment in wild-type mice

BM transplants were performed in wild-type female FVB mice as described for figure 2 except that 30mg/kg BADGE or control vehicle (DMSO 10%) were administered through daily intra-peritoneal injections from the day prior to irradiation until day 14 post-transplant. a.H&E stain of femurs from mice sacrificed on day 17 post-transplant, when the donor CD45.2 wildtype BM was recovered and purified by FACS. b.White blood cell (WBC) counts in peripheral blood on the post-transplant period show accelerated recovery in BADGE-treated mice. c.Colony forming unit assay (CFU) from the recovered donor BM