At birth, hematopoietic red marrow occupies virtually the entirety of the bone marrow space, but with age, non-hematopoietic fatty marrow gradually predominates 4
. This so-called “fatty degeneration” of the marrow is a dynamic and reversible process 5,6
. To investigate whether marrow adipocytes influence hematopoiesis, we surveyed the mouse skeleton for bones that harbour fatty marrow under normal conditions. We found that the spine of adult mice manifests a proximal to distal gradient of bone marrow adipocytes: thoracic vertebrae are virtually adipocyte-free, while vertebrae starting at the level of the third or fourth tail segments are highly adipocytic (). We isolated bone marrow from the thoracic and tail vertebrae of 12 week-old mice and quantified the hematopoetic stem cell and short-term progenitor compartments both phenotypically and functionally (schema in supplementary ). BM from tail vertebrae contains only 25% as many CD45+ hematopoietic cells per segment as thoracic BM, thus confirming the reduced overall hematopoietic cellularity evident by histology (, supplementary figure 2a
). Using flow cytometry, we determined the relative frequency of hematopoietic stem cells (HSC, ckit+Sca1+Lin-Flk2- or KLSF), multipotent progenitors (MPP), common myeloid progenitors (CMP), granulocyte-monocyte progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) in these different regions of the spine 10
. The percentage of all progenitor classes was reduced 2–3 fold in the CD45+ hematopoietic cells of adipocyte-rich BM of the tail vertebrae compared to non-adipocytic BM from the thoracic vertebrae (, supplementary figure 2b
). Congruent with the FACS phenotype data, long-term repopulating HSCs, short-term repopulating progenitors, spleen colonies and methylcellulose colony forming units were reduced 1.5–3 fold in adipocyte-rich BM from tail vertebrae as compared to adipocyte-free BM from the thoracic vertebrae (, supplementary figure 2c
). These phenomena are neither due to age nor weight-bearing status, for we observed a similar reduction in the frequency of short-term progenitors in younger (4 week-old) and older mice (13 months), as well as in another consistently fatty yet weight-bearing region of the mouse skeleton, the distal tibia (supplementary figures 3a–3f
). Reduced frequency of CMPs and primitive CFUs also accompany the increased BM adiposity of femurs from leptin deficient obese mice (supplementary figure 4a–4d
). Moreover, we found that BM-derived adipocytes reduce the expansion of hematopoietic cells in stromal transwell co-cultures, indicating that adipocytes release diffusible inhibitors of hematopoiesis (supplementary figure 5a–5d
). We therefore conclude that adipocyte-rich marrow is associated with lower absolute levels and relative numbers of hematopoietic progenitors.
Hematopoietic stem cells and progenitors are reduced in number, frequency and cycling capacity in adipocyte-rich bone marrow during homeostasis
To investigate the mechanism of reduced hematopoietic activity of adipocytic BM, we performed cell cycle analysis of the progenitor compartments. In all cases, we found fewer progenitors in the replicating phases of the cell cycle (S/G2/M) within the adipocyte-rich BM (, supplementary figure 6a
). Early progenitors (HSC/MPP) presented no significant difference in their G0
ratio, while late progenitors (GMP/CMP/MEP) presented a significant increase in the G0
ratio (supplementary figure 6b
). To determine if the slow-cycling HSCs within the tail BM were functional, as opposed to senescent, we FACS-sorted HSCs (ckit+Lin-Sca1+Flk2-, KLSF) and transplanted them competitively into lethally irradiated mice. We observed no difference in repopulating activity between HSCs from tail and thorax in the first month post-transplant. However, multilineage long-term engraftment was significantly higher in HSCs purified from tail BM (), suggesting that the slow-cycling progenitors in adipocytic tail BM are relatively quiescent and not senescent. This is consistent with the relative predominance of CD34low HSCs within the KLSF fraction of tail BM (), a phenotype associated with long–term repopulation activity 11
. Taken together, our data establish tail vertebrae as a model for the study of fatty marrow in the mouse, and demonstrate that adipocyte-rich marrow manifests altered hematopoiesis. HSCs and short-term progenitors are functionally reduced on a per cell basis in fatty marrow due to reduced cycling at the level of the HSC, MPP and CMP compartments.
To determine if the association between adipocytic marrow and reduced hematopoietic progenitor frequency is purely correlative, or whether adipocytes actively compromise hematopoiesis, we studied the lipoatrophic “fatless” A-ZIP/F1 mouse, which cannot form adipocytes due to the expression of a dominant negative form of C/EBP under the adipocyte fatty-acid binding protein 4 (aP2/FABP4) promoter 8
. In contrast to wildtype mice, we found that the absence of adipocytes in fatless A-ZIP/F1 mice rescued hematopoiesis in the tail, such that A-ZIP/F1 mice presented no significant difference in the frequency of CFUs from thorax or tail BM (supplementary figure 7a–b
), indicating that compromised osteogenesis due to the non-weight-bearing nature of these bones cannot explain the hematopoietic defect of wildtype, adipocyte-rich tail vertebrae 12
. Importantly, although fatless A-ZIP/F1 mice are diabetic, their blood counts were similar to controls during homeostasis, and their femoral BM showed no competitive advantage over BM from wild-type littermates, arguing that the diabetic milieu does not account for the observed alterations in the hematopoietic compartment (supplementary figures 8a–c
). We therefore conclude that the presence of adipocytes is necessary to observe reduced hematopoiesis in adipocyte-rich tail bone marrow.
We then analyzed the effect of adipocytes on hematopoietic recovery following bone marrow transplantation. Between the second and fourth week after lethal irradiation, the bone marrow space throughout the mouse skeleton becomes replaced by adipocytes. During this post-transplant period mice (and human patients) depend on fast cycling, short-term hematopoietic progenitors to rescue their otherwise lethal pancytopenia 13
. Given our prior data, we predicted that the compromised adipogenesis in the A-ZIP/F1 mouse would enhance hematopoietic recovery in the post-transplant period through increased proliferation of short-term progenitors. We transplanted wildtype BM (CD45.2) into either wildtype or fatless A-ZIP/F1 littermates (CD45.1, ). In contrast to control mice, A-ZIP/F1 fatless mice exposed to lethal doses of irradiation produced markedly fewer adipocytes in the marrow cavity (). We monitored leukocyte recovery in the post-transplant period and found that recovering A-ZIP/F1 fatless mice have up to 4 times higher leukocyte counts in peripheral blood relative to their wild-type controls (). We also observed significantly accelerated recovery in the hemoglobin content of peripheral blood (). Importantly, both wildtype and A-ZIP/F1 fatless recipients showed comparable high-level long-term donor chimerism after the primary transplant (supplementary figure 8d
). In the third week post-transplant, we recovered the donor CD45.2 BM from the adipocyte-rich wildtype or the adipocyte-free AZIP/F1 femurs. We found a pronounced increase in hematopoietic progenitors in the recovering CD45.2 BM isolated from fatless A-ZIP/F1 mice as determined by flow cytometry (), methylcellulose colony forming assays ( and supplementary figure 8e
) and short-term competitive repopulation into secondary recipients (). Collectively, these results indicate that the lack of adipogenesis in A-ZIP/F1 recipient mice enhances hematopoietic recovery after lethal irradiation by enhancing engraftment of short-term progenitors, and further supports the conclusion that adipocytes in fatty marrow hinder hematopoietic progenitor expansion.
The lack of bone marrow adipocytes post-irradiation in fatless mice enhances hematopoietic progenitor expansion and post-transplant recovery
During our studies, we observed that bone marrow ablation in lethally irradiated A-ZIP/F1 fatless mice was accompanied by marked osteogenesis. Trabecular bone was increased in the femurs of transplanted A-ZIP/F1 fatless mice compared to wild-type controls (), a phenomenon also apparent in the tail and in mice that were lethally ablated but received no transplant (supplementary figures 9a–c
). High-resolution micro-Computerized Tomography (mCT) confirmed a 5-fold increase in trabecular bone that was specific to fatless A-ZIP/F1 mice after BM transplantation (). Incorporation of 18
F measured by positron emission tomography (mPET) confirmed an increased bone metabolism, indicating new bone deposition in tails and tibias after bone marrow transplantation that was maximal in the second week post-transplant (). Our data show that simultaneous ablation of the hematopoietic and BM adipocyte compartment can induce osteogenesis, which, as shown by others, promotes a more supportive environment for hematopoietic reconstitution that could explain the positive effect of adipocyte ablation in BM engraftment 1, 2
. Our observation is compatible with a previous report that surgical removal of the fatty marrow in rabbit tibias induces transient hematopoietic infiltration and new osteoid and trabecular bone formation 14
. In addition to creating an osteogenic environment, fatless A-ZIP/F1 mice may accumulate mesenchymal elements that support hematopoietic recovery, or may be deficient in osteoclast elements that would antagonize trabecular bone growth during the recovery phase of lethal irradiation. Importantly, preventing the formation of BM adipocytes alone does not cause osteogenesis 15
, indicating that osteogenesis requires simultaneous ablation of both the adipocytic and hematopoietic compartments. These data suggest a three-way co-regulation of hematopoiesis, osteogenesis and adipogenesis within the BM compartment.
Ablation of the hematopoietic compartment in fatless A-ZIP/F1 mice during BM transplantation induces osteogenesis
Finally, we tested whether blocking adipogenesis pharmacologically could enhance bone marrow engraftment in wild-type mice. The PPARγ inhibitor Bisphenol-A-DiGlycidyl-Ether (BADGE) has been shown to prevent bone marrow adipocyte formation in vitro
and in vivo
in models of streptozotocin-induced diabetes 9, 15
. Importantly, BADGE does not enhance hematopoietic colony formation in vitro
, when BM cells are isolated from their stromal microenvironment (supplementary figure 10
). We administered BADGE to lethally irradiated mice for the two weeks following bone marrow transplantation, and observed successful inhibition of BM adipocyte formation (), higher peripheral blood leukocyte counts (), and an enrichment in colony forming units (). Our results demonstrate that the negative influence of adipocytes on post-transplant hematopoietic engraftment can be overcome pharmacologically, and suggest that PPARγ inhibitors, or other adipocyte inhibitors such as the novel aP2 inhibitor BMS309403 16
, might serve as adjuvants to hematopoietic recovery in clinical bone marrow transplantation.
Pharmacological inhibition of adipocyte formation enhances BM engraftment in wild-type mice
Collectively, our results contradict the classical dogma that adipocytes act as passive space fillers in the marrow. We demonstrate that adipocyte-rich marrow harbors a decreased frequency of progenitors and relatively quiescent stem cells. Moreover, we observe that mice that are genetically deficient in adipogenesis show accelerated hematopoietic recovery after bone marrow ablation, a phenomenon that can be reproduced pharmacologically in wild-type mice through PPARγ inhibition. These results suggest a novel therapeutic approach to enhance hematopoietic engraftment following marrow or cord blood transplantation, or to ameliorate aplasia in genetic bone marrow failure syndromes. Furthermore, our results suggest a plausible mechanism for the reports of myelosuppression17, 18, 19
in patients treated with the PPARγ agonist rosiglitazone, a diabetes drug known to increase marrow adiposity 20
Our data indicate a predominantly suppressive influence of adipocytes on hematopoiesis within the bone marrow microenvironment. BM adipocytes are less supportive of hematopoiesis in vitro
than their undifferentiated stromal or pre-adipocytic counterparts, in part due to reduced production of growth factors such as GM-CSF and G-CSF 21, 22
. Moreover, adipose tissue secretes neuropillin-1 23
, lipocalin 2 24, 25
, adiponectin 26
and TNFalpha 27, 28
, each of which can impair hematopoietic proliferation. Of note, TNFalpha and adiponectin inhibit progenitor activity while positively influencing the most primitive HSCs 27, 29
, suggesting that adipocytes prevent hematopoietic progenitor expansion while preserving the hematopoietic stem cell pool. Adipocytes and osteoblasts originate from mesenchymal stem cells within the bone marrow, where both compartments hold a reciprocal relationship 30
. Balancing the supportive role of the osteoblast in the HSC niche, our data implicate adipocytes as negative regulators of hematopoiesis. Further studies will address the molecular players involved in the hematopoietic inhibition imposed by fatty marrow.