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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.

Kuznetsov SA, Riminucci M, Ziran N, Tsutsui TW, Corsi A, Calvi L, Kronenberg HM, Schipani E, Robey PG, Bianco P - J. Cell Biol. (2004)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

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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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.
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fig6: 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.

Mentions: In primary culture, tg strains demonstrated a shorter doubling time than wt strains (32.2 vs. 35.5 h, respectively). Over 17 d in culture, tg and wt strains underwent 12.6 and 11.5 population doublings, respectively. At the end of ex vivo expansion, total cell yield per initially plated CFU-F was 2.3-fold higher for tg strains than for wt strains (6.8 × 103 vs. 2.9 × 103, respectively). The frequency of CFU-Fs at the end of culture (before in vivo transplantation) was assessed by enumerating the number of CFU-Fs, which was done by plating 103 cells per strain in 25-cm2 flasks (CFEtN). At this time, the frequency of CFU-Fs in the total population had become significantly higher (more than twofold) in tg strains than in wt strains (Fig. 6 b). The total numbers of CFU-Fs in wt and tg strains at the end of culture were then determined based on CFEtN and total cell yield, and ratioed to the total number of explanted CFU-Fs in each strain (CFU-FtN/CFU-Ft0). This revealed a mean 52.7-fold increase in CFU-Fs in wt strains, and an average 290.2-fold increase in CFU-Fs in tg strains (an approximately sixfold increase in tg strains compared with wt strains; Fig. 6 c). Hence, threefold as many total cells, but approximately sixfold as many CFU-Fs per single, originally explanted CFU-F, were obtained for tg strains compared with wt strains over the same time in culture. The CFU-Fs from tg mice expanded in culture to a greater extent, and generated more CFU-Fs per total number of stromal cells, than those from wt mice.


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.

Kuznetsov SA, Riminucci M, Ziran N, Tsutsui TW, Corsi A, Calvi L, Kronenberg HM, Schipani E, Robey PG, Bianco P - J. Cell Biol. (2004)

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.
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Related In: Results  -  Collection

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fig6: 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.
Mentions: In primary culture, tg strains demonstrated a shorter doubling time than wt strains (32.2 vs. 35.5 h, respectively). Over 17 d in culture, tg and wt strains underwent 12.6 and 11.5 population doublings, respectively. At the end of ex vivo expansion, total cell yield per initially plated CFU-F was 2.3-fold higher for tg strains than for wt strains (6.8 × 103 vs. 2.9 × 103, respectively). The frequency of CFU-Fs at the end of culture (before in vivo transplantation) was assessed by enumerating the number of CFU-Fs, which was done by plating 103 cells per strain in 25-cm2 flasks (CFEtN). At this time, the frequency of CFU-Fs in the total population had become significantly higher (more than twofold) in tg strains than in wt strains (Fig. 6 b). The total numbers of CFU-Fs in wt and tg strains at the end of culture were then determined based on CFEtN and total cell yield, and ratioed to the total number of explanted CFU-Fs in each strain (CFU-FtN/CFU-Ft0). This revealed a mean 52.7-fold increase in CFU-Fs in wt strains, and an average 290.2-fold increase in CFU-Fs in tg strains (an approximately sixfold increase in tg strains compared with wt strains; Fig. 6 c). Hence, threefold as many total cells, but approximately sixfold as many CFU-Fs per single, originally explanted CFU-F, were obtained for tg strains compared with wt strains over the same time in culture. The CFU-Fs from tg mice expanded in culture to a greater extent, and generated more CFU-Fs per total number of stromal cells, than those from wt mice.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

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.

Show MeSH