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. 2004 Dec 20;167(6):1113-22.
doi: 10.1083/jcb.200408079.

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow

Sergei A Kuznetsov  1 , Mara Riminucci, Navid Ziran, Takeo W Tsutsui, Alessandro Corsi, Laura Calvi, Henry M Kronenberg, Ernestina Schipani, Pamela Gehron Robey, Paolo Bianco
Affiliations

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow

Sergei A Kuznetsov et al. J Cell Biol. .

Abstract

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.

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Figures

Figure 1.
Figure 1.
Impaired development of the marrow cavity and hematopoietic tissue in long bones of COL1-caPPR mice. (a) High resolution radiograms of the femurs of wt and tg mice at 2 wk. Note the marked difference in length of the marrow cavity (arrows). (b) High resolution radiograms of tibiae and fibulae of wt and tg mice at 2 wk. Note the different lengths of the primary spongiosa (arrows). (c and d) Histological sections of the distal metaphysis of the femur (c) and proximal metaphysis of the tibia (d) at 2 wk. Red marrow extends to the metaphyseal end of the primary spongiosa in wt mice (arrows), but only medullary bone is present in tg mice. (e and f) High resolution contact microradiography (e) and microCT analysis (f) of tibiae at 2 wk. The excess medullary bone formed in tg mice is obvious with both techniques. MicroCT demonstrates that the normal partition between cortical bone and marrow space is lost in tg mice, and that both are replaced by a continuous plexus of cancellous bone. Sections extend from 0.5 to 2.5 mm below the physis.
Figure 2.
Figure 2.
Impaired development of adipose (yellow) marrow in COL1-caPPR mice. Histology of the calcaneum at 2 wk and 3 mo. At 2 wk, the wt marrow cavity contains red hematopoietic marrow (a) with scattered adipocytes, and frequent multivacuolar, developing adipocytes (c and inset). In tg mice, a distinct cavity is not observed, hematopoiesis and adipocytes are absent, and an excess of bone is present (b and d). At 3 mo, hematopoiesis is no longer present, and the marrow cavity is filled with mature adipocytes in wt mice (e). No cavity is present in tg mice, and only rare adipocytes are found in narrow vascular spaces interrupting the continuity of the excess bone (f).
Figure 3.
Figure 3.
Relative growth of marrow cavities with respect to bone over time. High resolution radiograms of tibia and fibula at 1, 3, and 5 mo. At 1 mo, a much longer primary spongiosa is observed in tg mice than in wt mice. In continuity with the extended primary spongiosa, abundant trabeculae are detected in the medullary cavity of the diaphysis. Corresponding regions in wt tibia show a bone-free marrow cavity. At 3 mo, a dense primary spongiosa is still present in the tg tibia, albeit much reduced in length compared with what is observed at 1 mo. Below this region, a marrow cavity is discernible. However, it is occupied by a dense meshwork of bone trabeculae. Corresponding regions in the wt tibia show a bone-free marrow cavity. A similar pattern is observed at 5 mo. Asterisks mark corresponding regions in the primary spongiosa, metaphysis, and diaphysis in radiograms of wt and tg mice.
Figure 4.
Figure 4.
Transient fibrous dysplastic phase in bone and marrow of Col1-PPR mice. (a and b) Transmitted light views of standard H&E-stained sections of the proximal metaphysis of the tibia at 2 wk and 3 mo. The medullary bone is lined by typical cuboidal osteoblasts (ob) at 2 wk (a), and, although excessive, it is otherwise histologically normal. In contrast, morphologically typical osteoblasts cannot be recognized, and only spindle-shaped cells (sc) fill the spaces between trabeculae at 3 mo (b). The orientation of trabeculae is more haphazard, giving the tissue a dysplastic appearance. (c and d) Fluorescence microscopy images of the same sections. No cement lines (marks of bone remodeling events) are seen at 2 wk (c), which is consistent with the primary nature of the medullary bone. A complex pattern of cement lines (which remain nonfluorescent in H&E-stained sections) is seen at 3 mo (d, arrows), testifying to the occurrence of multiple remodeling cycles.
Figure 5.
Figure 5.
Histology of the bone/marrow organ and CFU-F frequency in mice at skeletal maturity. (a) Histology of the proximal metaphysis of the tibia in wt and tg mice at 4.5 mo. Hematopoietic marrow now fills the marrow cavity up to the physis in both wt and tg mice. A marked excess of trabecular bone is observed in tg mice compared with in wt mice (undecalcified MMA sections, von Kossa staining). (b) Representative primary cultures of cells established at clonal density from wt and tg mice. Note the higher number of colonies (CFU-F) in wt cultures.
Figure 6.
Figure 6.
Frequency and expansion capabilities of CFU-F. (top) Frequency of CFU-F at the time of explantation (t0). Results shown as number of colonies per 105 nucleated bone marrow cells (mean of triplicate determinations per mouse). The tg marrow is reduced in CFU-F, relative to the wt marrow (analysis of variance [ANOVA], Scheffe's F test 309.16* [significant at 95%], P < 0.0001). (middle) Frequency of CFU-F at the end of ex vivo expansion (day 17, tN). Results shown as number of colonies per 103 stromal cells (mean of triplicate determinations per two cell strains per genotype). Strains of tg mice are enriched in total CFU-F, relative to strains of wt mice (ANOVA, Scheffe's F test 94.03* [significant at 95%], P < 0.0002). (bottom) Fold increase in CFU-F over the culture period was calculated as the ratio of the total number of CFU-Fs at the end of the culture period to the total number of CFU-Fs in the marrow explants. Results based on triplicate determinations in duplicate experiments (ANOVA, Scheffe's F test 124.52* [significant at 95%], P = 0.02). (top, middle, and bottom) Error bars indicate SD of the mean.
Figure 7.
Figure 7.
Osteogenic potential of stromal strains. (a) Histology of ectopic ossicles formed subcutaneously in immunocompromised mice by strains derived from wt and tg mice transplanted with an osteoconductive carrier (HA/TCP particles; HA). Formation of bone (b) and marrow (bm) is obvious in transplants of wt cells at 28, 42, and 56 d. Adipocytes in the hematopoietic marrow are evident at 42 and 56 d. Formation of bone, but not of red marrow, is obvious in transplants of tg cells at 28, 42, and 56 d. Adipocytes are not formed, and only fibrous tissue (ft) fills the spaces between bone surfaces. (b) Histomorphometric assessment of the amount of bone (bone volume/total volume; BV/TV %) formed in ectopic ossicles did not reveal differences between wt and tg strains at any time point. A similar increase in the amount of bone was observed at 56 d compared with earlier time points in wt and tg transplants. Error bars indicate SD of the mean.
Figure 8.
Figure 8.
Gross, radiographic, and histological analysis of subcutaneous transplants of stromal strains in conjunction with collagen sponges. Gross appearance of the transplants generated by wt (a and e) and tg (c and g) strains harvested at 42 (a and c) and 56 d (e and g). Hematopoiesis gives a red color to wt transplants (a and e), whereas the collagen sponge remains pale gray in spite of vascularization (well apparent in c) in tg transplants (c and g). High resolution radiograms of the same structures for wt (b and f) and tg (d and h) transplants. Bone formation is readily detected in wt transplants (b and f), and even a distinct bony cortex is easily resolved in the ectopic ossicles. No bone was formed by tg cells (d and h). (i and j) Nondecalcified sections stained with Goldner's trichrome stain. (k–n) Paraffin sections stained with H&E. (i–n) Examination of wt transplants at 28 (i), 42 (k), and 56 d (m) revealed the progressive formation of a complete heterotopic ossicle, including a bony cortex and a medullary cavity with trabecular bone (*), and the establishment of a complete hematopoietic marrow. Fully mature adipocytes (#) are well apparent by day 42. Neither bone nor a hematopoietic marrow was present at any time point in tg transplants (j, l, and n).
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