Bone marrow OPN production is altered by PTHR activation on osteoblasts
To validate that OPN is produced in a modulated manner at sites relevant for hematopoiesis, we performed immunohistochemistry on tibia sections either from wild-type animals or from animals with the activation of the PTHR. To focus specifically on the osteoblast production of OPN, we used mice transgenic for a constitutively active PTHR driven by the osteoblast-specific collagenα1(I) promoter. Production of OPN in the marrow cavity under normal homeostatic conditions was generally in immediate proximity to spindle-shaped osteoblasts lining trabecular bone surfaces (). In contrast, with activated PTHR, OPN staining was markedly increased and extended diffusely from the trabecular surface into the interstitium surrounding hematopoietic cells (). We previously demonstrated that osteoblasts were producing this increased OPN using a combination of in situ hybridization and immunohistochemistry (23
). However, we noted that other hematopoietic cells can also express OPN in response to cytokine stimuli (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20041992/DC1
). Collectively, these data indicate that OPN is produced in varying amounts with the resulting varied distribution affected by cell stimulation. Our demonstration of varied OPN production by the known niche constituent, the osteoblast, provides support to further investigate the role of OPN in bone marrow homeostasis.
Figure 1. OPN is increased in bone marrow with the activation of osteoblasts. Immunohistochemistry of tibia sections from wild-type (left) or littermate transgenic (right) mice with a constitutively activated PTH/parathyroid related peptide receptor driven by a (more ...)
Expanded primitive cell pool in OPN-deficient mice
Initially, we characterized the bone marrow hematopoietic compartment under steady-state conditions using animals engineered to be deficient in OPN or their wild-type littermates as controls (15
). The total cellularity (OPN+/+
, 54.4 ± 4.7 × 106
cells and OPN−/−
, 51.4 ± 3.8 × 106
cells; P = 0.31, n
= 9) and the proportion of differentiated cells such as B- and T-lymphocytes, granulocytes, or erythroid cells were not altered in the absence of OPN ( B). Therefore, OPN deficiency has minimal impact on the steady state of more mature blood elements and similarly modest changes in precursor populations as determined by quantitating cells without mature lineage markers (lin−
; absolute numbers: OPN+/+
, 2.6 × 106
± 0.2; and OPN−/−
, 3.0 × 106
± 0.3 per femur; P = 0.16, n
= 8) or with markers of differentiating erythroblasts (Ter119/CD71) or B cells (B220/IgM−
; Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20041992/DC1
). However, flow cytometric analyses revealed significantly more primitive cells in the stem cell–enriched Sca1+
cells in OPN-deficient mice compared with controls (OPN+/+
, 1.44 ± 0.26% vs. OPN−/−
, 2.64 ± 0.58%; P = 0.03, n
= 8) (absolute number: 2.92 ± 0.55 × 104
vs. 4.68 ± 1.12 × 104
per femur pair; P = 0.02, n
= 8; C; reference 24
). Within the Sca1+
population, the CD34−
subset has been defined to further purify cells capable of long-term reconstitution; we found that these cells were also considerably increased in the OPN-deficient animals (P = 0.02, n
= 8; D; reference 25
Figure 2. Primitive hematopoietic cells are increased in the bone marrow of OPN−/− mice, whereas mature cells are not. (A) Bone marrow cells of OPN+/+ (littermate control) and OPN−/− mice were harvested, counted, and stained with (more ...)
To assess the impact on cells defined by function, we initially performed colony assays using the methylcellulose colony-forming cell (CFC) assay for progenitors. A significantly lower number of CFCs in the bone marrow of OPN−/− mice were noted (OPN+/+, 30.6 ± 4.1 vs. OPN−/−, 19.05 ± 2.9 colonies per 104 bone marrow cells; P = 0.025, n = 5). As a measure of more primitive cells, we performed long-term cultures (LTCs) on primary murine bone marrow stroma using a limiting dilution LTC–initiating cell (IC) assay. OPN−/− bone marrow cells gave rise to a significantly higher number of LTC-ICs (P = 0.01, n = 5; E). Notably, the OPN-null cells were able to mature into normal-appearing colonies on wild-type stroma used in these assays, which suggests that OPN deficiency did not intrinsically impair hematopoietic cell differentiation.
To more accurately assess the impact of OPN on the stem cell compartment, we admixed cells in a 1:1 ratio from the wild-type and null genotypes and transplanted them into lethally irradiated wild-type recipients. 12 wk after transplantation, the relative abundance of each genotype was quantitated, and the OPN−/− cells represented 67.1 ± 1.6% (n = 8) of the bone marrow and blood cells ( F). The difference between the relative engraftment of OPN−/− to wild-type cells was highly statistically significant (P = 0.00001) and reflected an approximately twofold excess of stem cells present in the OPN−/− donor marrow. Proliferation, apoptosis, or other stem cell–autonomous effects could all account for these results and were subsequently addressed.
Transplantation analysis demonstrates a stroma-determined effect by OPN on hematopoietic stem cells
To address whether the impact of OPN was stem cell autonomous or stroma dependent, we performed sequential bone marrow transplantation, reasoning that a stem cell–autonomous effect would be retained with each transplant, whereas a nonautonomous or stroma-determined effect would not. Bone marrow from OPN+/+ or OPN−/− male animals (Ly5.2) was transplanted into lethally irradiated female Ly5.1+ mice. 2 mo after engraftment, 4–8 × 106 bone marrow cells were used as donor cells and again transplanted into new lethally irradiated Ly5.1+ recipients. After another 3-mo period, the bone marrows of the secondary recipients were analyzed. There was no difference in the total bone marrow cellularity of animals serially transplanted with OPN−/− or OPN+/+ bone marrow cells. Similarly, there was no difference in either the proportion or absolute number of the stem cell–enriched Sca1+kit+lin− fraction of Ly5.2+ cells in the bone marrow of animals serially transplanted with OPN−/− or OPN+/+ cells, suggesting the unaltered self-renewal ability of OPN-deficient stem cells ( A). To more accurately quantify the progenitor and primitive cell frequency in the bone marrow of the serially transplanted animals, we performed CFC and LTC-IC assays. We could not detect any notable differences between genotypes in either population as reflected by these assays (unpublished data). These data demonstrate that the alteration in primitive hematopoiesis (increased LTC-ICs and decreased CFCs) seen in an OPN-deficient animal was not persistent when cells from that animal were transplanted into a wild-type background. Why the cell numbers would revert back to a level resembling wild-type animals has several possible explanations. It is possible that OPN−/− stem cells do not home as well as OPN+/+ cells, meaning that fewer cells arrive at their supportive niche, which accounts for the result.
Figure 3. OPN−/− hematopoietic stem cell increase is not cell autonomous, but stroma dependent. (A) In a serial transplantation experiment using C57BL/6 wild-type mice (Ly5.1) as recipients for either OPN−/− or OPN+/+ bone marrow (more ...)
To directly address the issue of abnormal homing of seeding, we performed in vivo homing assays. Bone marrow cells of OPN+/+
(Ly5.2) mice were transplanted into lethally irradiated wild-type recipients (Ly5.1; 2 × 107
per animal). 14 h after transplantation, the recipient animals were killed and the bone marrow was analyzed by flow cytometry using the surface markers Ly5.1 and Ly5.2 simultaneously with stem cell markers. The proportion of donor cells (Ly5.2) was similar in the bone marrow of animals transplanted with OPN+/+
bone marrow ([OPN+/+
] 3.37 ± 0.4% vs. [OPN−/−
] 2.66 ± 0.2%; P = 0.08, n
= 3). However, the proportion of Sca1+
cells, a more primitive subset (26
), was twofold higher in the animals transplanted with OPN−/−
bone marrow compared with the controls ([OPN+/+
] 1.03 ± 0.1% vs. [OPN−/−
] 2.13 ± 0.1%; P = 0.001, n
= 3; B), which reflected the twofold higher proportion of stem cells in the bone marrow of the OPN−/−
donor animals before transplantation. Therefore, OPN-deficient stem cells do not appear to have any disadvantage in seeding or short-term (14 h) retention in the bone marrow.
To assess the possible role of the microenvironment itself in governing stem cell pool size, we cultivated stroma from either OPN+/+ or OPN−/− mouse bone marrow. Sca-1+ lin− mononuclear bone marrow cells from either genotype were then plated at limiting dilutions in standard LTC-IC conditions. The OPN−/− stroma was more capable of supporting LTC-ICs than wild-type stroma (365.5 ± 60.2 LTC-ICs/100,000 cells vs. 450.4 ± 63.1 LTC-ICs/100,000 cells; P = 0.002, n = 7; C). These data suggested that stroma was the determinant of primitive pool size and not the primitive cells themselves. This nonautonomous effect on primitive cells supported a role for OPN in the regulatory microenvironment and we further investigated that role.
To test the in vivo effects of the OPN−/− stroma, we transplanted wild-type cells into lethally irradiated OPN−/− or OPN+/+ animals. 12 wk after engraftment, the relative abundance of donor cells was examined by flow cytometry and functional LTC-IC assays. Marrow that had been engrafted in the OPN-deficient hosts demonstrated a statistically significant increase in phenotypic Ly5.2 Sca1+c-kit+lin− cells and functional LTC-ICs (4.72 ± 0.11 vs. 5.63 ± 0.49% of Sca1+c-kit+ cells in the lin− fraction, P = 0.049, n = 4; 0.59 ± 0.08 vs. 1.22 ± 0.26 LTC-ICs/100,000 cells, P = 0.049, n = 4; ) closely resembling the OPN-null phenotype. Therefore, the microenvironment provided by the OPN-deficient animals was able to support a greater number of primitive cells in a stroma-dependent manner. These data support the stem cell–nonautonomous nature of the OPN−/− effect.
OPN deficiency does not affect cell cycle kinetics, but alters stromal Jagged1 and Angiopoietin-1 expression and primitive cell apoptosis
To assess potential mechanisms by which the microenvironment of the OPN-deficient animals contributed to the expanded stem cell pool in OPN−/− mice, we assessed cell cycle kinetics. Bone marrow cells were stained with Sca1, c-kit, and lineage markers, and the cell cycle status was analyzed by simultaneous staining with the DNA dye Hoechst 33342. We observed a similar G0/G1 and S+G2/M percentage of Sca1+c-kit+lin− in the bone marrow of OPN+/+ and OPN−/− animals (S+G2/M OPN+/+, 0.22 and OPN−/−, 0.22%; pooled bone marrow of three animals each; A). These data indicate an unperturbed cell cycle status of primitive cells in the absence of OPN even though it is recognized that they cannot define the interval spent in any phase in a single cycle, nor the rapidity of cycling. To better address the latter issue, BrdU labeling was performed by exposing the animals to BrdU in their drinking water for variable intervals and examining the extent of BrdU uptake in primitive subsets of marrow cells by flow cytometry. Modest differences that did not achieve significance were noted between the genotypes at 3, 6, and 10 d ( B).
Figure 4. OPN−/− bone marrow has unaltered cell cycle profiles associated with increased stromal Jagged1 and Angiopoietin-1 expression and reduced primitive cell apoptosis. (A) Bone marrow Sca1+c-kit+lin− cells with a bright staining for (more ...)
Stem cell expansion may occur without increased proliferation in the context of Notch1 activation where stem cell self-renewal is favored over differentiation (27
). Activation of Notch1 on primitive hematopoietic cells in vivo was previously shown by us to result in an increase in primitive cells, but reduced progenitor cells; a similar phenotype was observed here (27
). Also, a link between Notch1 and OPN was reported by Iwata et al., who showed that OPN can reduce Notch1 receptor abundance on human CD34+
). Because the Notch1 ligand, Jagged1, has been shown to be produced by osteoblasts in the hematopoietic stem cell niche and to affect stem cell pool size (4
), we assessed the Jagged1 expression in marrow stromal cells. An increase in Jagged1 was observed in the OPN-deficient animals relative to wild-type controls (P = 0.02, n
= 6; C). To determine whether the reciprocal was true—that OPN stimulation of wild-type cells might decrease Jagged1—we exposed marrow stroma to OPN ex vivo for 4 h. Jagged1 was found to be significantly reduced statistically by OPN (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20041992/DC1
). Other molecular features of the stem cell niche recently defined include N-cadherin (3
) and Angiopoietin-1 (30
). We also examined the expression of these in stroma and noted modest, insignificant increases in N-cadherin (P = 0.08, n
= 6), but a more pronounced increase in Angiopoietin-1 in the absence of OPN (P = 0.02, n
= 6; C). Angiopoietin-1 has been defined as a molecule that can increase stem cells not by increasing proliferation, but rather by enhancing quiescence. These data suggest that the impact of OPN expression is likely multifaceted; it alters features of the niche that in combination change its capacity to nurture primitive hematopoietic cells, none of which are associated with increased proliferation.
An additional possible mechanism for increasing stem cell numbers without altering cycling kinetics is decreased cell death. To evaluate this, bone marrow cells were stained with stem cell markers and simultaneously with Annexin V and the DNA dye 7-AAD to determine the fraction of apoptotic cells indicated by the phenotype Annexin V+7-AAD−. We could detect a trend toward fewer apoptotic cells in the Sca1+c-kit+lin− bone marrow stem cell–enriched population in OPN−/− mice in comparison with controls (n = 4). Additionally, the OPN-deficient bone marrow in serially transplanted animals showed a lower fraction of apoptotic cells in the Sca1+c-kit+lin− cell population in comparison with controls, suggesting a preserved lower tendency of OPN-deficient stem cells to become apoptotic. Furthermore, we transplanted wild-type bone marrow into either the wild type or OPN-deficient recipients and noted that lineage-negative hematopoietic cells of the OPN+/+ genotype acquired a decreased apoptosis fraction similar to the OPN-deficient animal ( D), demonstrating that the basis for the change in apoptosis was stroma dependent. These results suggest that the enlarged stem cell pool in OPN-deficient mice may be caused, in part, by enhanced survival, but required further definition.
Soluble OPN reduces LTC-ICs and increases the apoptotic fraction of wild-type cells
We used exogenous OPN to assess its potential role in regulating primitive cells directly rather than through the altered expression of other regulators within the niche. Initial experiments used Sca1+lin− bone marrow cells of C57BL/6 mice cultured in a medium containing stem cell factor (SCF), Flt-3, TPO, and IL-3 with and without OPN for 7 d, after which the cells were counted and analyzed in functional in vitro progenitor and stem cell assays. The addition of soluble OPN led to a lower total cell number with an unperturbed absolute number of CFCs representing hematopoietic progenitor cell activity (n = 5; A). However, exogenous OPN led to a significantly lower absolute number of LTC-ICs (without OPN, 35.9 ± 5.14 LTC-ICs/well; with OPN, 16.41 ± 4.5 LTC-ICs/well; P = 0.002, n = 5; B).
Figure 5. Soluble OPN induces apoptosis of primitive hematopoietic cells. Sca1+lin− cells were isolated from the bone marrow of C57BL/6 mice and cultured in IMDM containing 10% FCS, SCF, Flt-3, TPO, and IL-3 with or without 1 μg/ml OPN. After 7 (more ...)
We next analyzed the fraction of apoptotic cells by staining with lineage markers, 7-AAD and Annexin V, and detected a higher percentage of Annexin V+7-AAD− cells in the lin− cell population cultured with OPN, which was consistent with increased apoptosis ( C). A similar effect was seen with Sca+ lin− cells in the OPN−/− animals and was neutralized with anti–OPN-specific antibody (unpublished data). Therefore, the addition of OPN documented an effect on primitive cell apoptosis that had been suggested by the analysis of the OPN-deficient mice in vivo. OPN exerts a proapoptotic effect on primitive cells, potentially constraining the size of the stem cell pool.
OPN restricts primitive cell expansion induced by osteoblast activation
To determine whether OPN acts to limit the dimensions of the stem cell pool under conditions in which stem cell expansion occurs, we took advantage of a previously reported in vivo context. PTH is capable of activating niche osteoblasts and, in a Notch-mediated manner, expand the number of stem cells in vitro and in vivo (4
). PTH has been shown to be physiologically increased in settings such as myelotoxic ablation with radiation and chemotherapy (31
). Stimulation with PTH increases OPN production, so we hypothesized that the degree of stem cell expansion possible by PTH niche activation may be restricted by OPN. Using an OPN-null or wild-type mouse, we assessed the number of primitive cells after 4 wk of PTH stimulation. There was the expected difference in the number of Sca1+
between the OPN-null and wild-type mouse before PTH (). With PTH treatment, there was an increase in the Sca1+
cells in each genotypic background. The magnitude of Sca1+
increase induced by PTH was greater in both proportion and absolute number in the null animals (10.0 vs. 7.5%, or 5.94 × 104
vs. 3.82 × 104
stem cells per femur pair). These data indicate that activation of the niche can increase primitive cells to a greater degree without OPN present in the milieu, arguing that OPN limits the degree of primitive cell increase that can be attained with the stimulation of osteoblasts.
Figure 6. OPN deficiency permits increased primitive hematopoietic cell compartment expansion after niche activation by PTH. OPN+/+ and OPN−/− mice were treated with PTH by daily injection for 4 wk. The bone marrow was analyzed by flow cytometry. (more ...)