Parabiosis model does not show presence of circulating osteoprogenitor cells.

The goal of this study was to determine the presence of osteoprogenitor cells in the peripheral blood. Experiments were conducted with a parabiosis model in which osteoblast specific transgenic mice (Col2.3GFP or hOC-GFP) were surgically joined with a transgenic mouse where herpes virus thymidine kinase gene is under the control of the collagen alpha1 promoter (Col2.3DeltaTK). This method permits conditional ablation of osteoblasts by ganciclovir (GCV) treatment. In parabionts treated with GCV for 15 days or 1.5-2 months, GFP (hOC-GFP or Col2.3GFP) expression was not detected in histological preparations or in marrow stromal cell cultures from the Col2.3DeltaTK parabiont. Finally, Col2.3GFP/Col2.3DeltaTK pairs were treated with GCV for 15 days and allowed to recover from GCV for 3 months. Again there was a failure to detect Col2.3GFP expressing cells in the Col2.3DeltaTK parabiont. These observations, at least within the limits of this model system, allow the conclusion that osteoprogenitor cells do not readily circulate.

Fibroblasts/myofibroblasts that participate in cutaneous wound healing are not derived from circulating progenitor cells.

Dermal fibroblasts/myofibroblasts involved in the wound healing are thought to originate from the resident fibroblast progenitors. To test the hypothesis of an extra dermal origin of the dermal fibroblasts/myofibroblasts, bone marrow (BM) transplantation and parabiosis experiments were initiated utilizing a collagen promoter green fluorescent protein (GFP) reporter transgene as a visible marker for dermal fibroblasts/myofibroblasts. BM transplantation experiments using BM from Col3.6GFPsapph transgenic mice showed no evidence that BM derived progenitors differentiated into dermal fibroblasts/myofibroblasts at the wound site. Rather the GFP positive cells (GFP+) observed at the wound site were not dermal fibroblasts/myofibroblasts but immune cells. These GFP+ cells were also detected in the lung and spleen. Furthermore, GFP+ fibroblasts were not detected in primary dermal fibroblast cultures initiated from BM chimeras. Using the same transgenic mice, parabiotic pairs were generated. One partner in the parabiosis carried a GFP expressing transgene while the other partner was a non-transgenic C57BL/6 mouse. Similar to the BM transplantation experiments, GFP+ immune cells were detected in the wound of the non-transgenic parabiont, however, GFP expressing dermal fibroblasts/myofibroblasts were not observed. Collectively, these data suggest that dermal fibroblast/myofibroblast progenitors do not readily circulate. The expression of the Col3.6GFPsapph in the hematopoietic cells confirmed that our methods were sensitive enough to detect Col3.6GFP expressing dermal fibroblasts derived from the peripheral circulation if they had originated in the BM.

Parabiosis and transplantation models show no evidence of circulating dermal fibroblast progenitors in bleomycin-induced skin fibrosis.

To test the hypothesis of an extra-dermal origin of dermal fibroblasts, parabiosis, and transplantation models were developed utilizing a collagen promoter green fluorescent protein (GFP) reporter transgene expressed in dermal fibroblasts. Parabiotic pairs were treated with bleomycin to induce the skin fibrosis that was evaluated for a dense deposition of collagen and inflammatory cell infiltrates in the thickened dermis in comparison with parabiotic pairs treated with saline. Although, in all cases, repeated injection of bleomycin for 4 weeks induced skin fibrosis, only a few GFP positive cells were detected in skin samples from some of the treated non-transgenic mice. Unexpectedly, similar results were observed in saline treated controls. Furthermore, bone marrow chimeras were created in which non-transgenic recipient mice received injections of bone marrow cell preparations isolated from pOBCol3.6GFP transgenic mice. After bone marrow chimerism had been successfully established, fibrotic lesions in the skin were induced by local bleomycin injections. Donor GFP expressing cells were observed in the skin from all recipient mice. However, no difference in the presence of GFP expressing cells was observed between non-treated mice or mice treated with bleomycin or saline. A large number of GFP expressing cells were observed in the lung preparations from all chimeric mice. Mac-3 antibody immunostaining confirmed a macrophage phenotype for these GFP expressing cells suggesting the expression of the pOBCol3.6GFP transgene in a non-collagen producing cell. Based on these observations, we found no evidence of circulating dermal fibroblast progenitors that participate in the development of bleomycin-induced skin fibrosis.

Regulation of collagen gene expression in the Tsk2 mouse.

The tight skin 2 (Tsk2) mutation is an ENU induced dominant mutation localized on mouse chromosome 1. While the molecular defect is unknown, Tsk2/+ mice display cutaneous thickening associated with excessive matrix production and are used as a model of scleroderma. The purpose of this study was to examine the cellular mechanisms associated with the excessive synthesis of matrix macromolecules using a collagen promoter GFP reporter transgene (pOBCol3.6GFP) as a marker of Col1a1 expression. This analysis of pOBCol3.6GFP expression in Tsk2/+ skin showed an increase in transgene activity compared to wild-type (+/+) samples. In addition, an increased area of "high" GFP fluorescence in Tsk2/+ dermis in both 1- and 4-month-old mice was observed that was also associated with an increased number of dermal fibroblasts per unit area of dermis. These data collectively suggest an important mechanism of Tsk2/+ skin fibrosis; an increased number of collagen expressing cells as well as elevated collagen expression on a per cell basis. During this study it was noted that Tsk2/+ mice appeared consistently smaller than wild-type (+/+) siblings and measurements of body length revealed a decrease (5-10%) in 1- and 2-month-old Tsk2/+ mice as well as a decrease in body weight in both age groups as compared to wild-type (+/+) control mice. Femur length was also decreased (2-9%) in Tsk2/+ mice. Finally, in contrast to Tsk/+ mice that display an emphysema-like lung pathology, histological sections of lungs from Tsk2/+ mice were normal and indistinguishable from wild-type (+/+) controls.

Marfan-like skeletal phenotype in the tight skin (Tsk) mouse.

Tight skin (Tsk) is an autosomal dominant mutation located on mouse chromosome 2 and is associated with an intragenic duplication of the fibrillin 1 (Fbn1) gene. Mutant mice (Tsk/+) display a tightness of skin in the interscapular region, lung emphysema, myocardial hypertrophy, skeletal overgrowth, and kyphosis. It is hypothesized in this study that in Tsk mice the mutation in Fbn1 alters bone cell metabolism. A detailed study of the Tsk skeletal phenotype revealed that Tsk mice have significantly longer femurs and axial skeleton as well as vertebral abnormalities. Cortical and trabecular bone volumes were significantly decreased in Tsk femurs from 2- and 4-month-old mice (13% and 39%, respectively) as well as trabecular thickness, number, connectivity, and surface area. These skeletal differences were also associated with a reduction in bone mineral density in mutant mice. Expression of the osteoblast-specific genes Col1a1, BSP and OC was examined in marrow stromal cell cultures at various time points. A decrease in the rate of maturation of the Tsk cells was indicated by a delay in the appearance of OC expression. These initial experiments demonstrated a significant role of the fibrillin 1 protein in the extracellular matrix of bone cells.

The 3.6 kb DNA fragment from the rat Col1a1 gene promoter drives the expression of genes in both osteoblast and osteoclast lineage cells.

The type I collagen promoter has been used to develop transgenic constructs that are able to mark different stages of osteoblastic differentiation. The pOBCol3.6 promoter is active in early mesenchymal progenitors, including preosteoblasts and osteoblasts, while the pOBCol2.3 promoter is more restricted, showing expression in mature osteoblasts and osteocytes. Transgenic mouse lines have been created that express various GFP reporters under the control of both promoters. These transgenic mice permit the tracking of osteoblastic lineage progression in vitro. They also represent a system to test lineage progression in vivo after the transplantation of progenitors. A parabiosis system was used in which pOBCol3.6GFP transgenic mice were surgically joined with mice bearing a Col2.3DeltaTK transgene. The Col2.3DeltaTK transgenic mouse bears a herpes thymidine kinase gene driven by the pOBCol2.3 promoter, and upon treatment with gancyclovir (GCV) displays extensive destruction of the bone lining cells. After a common circulation was established, parabiotic pairs were treated with GCV for 15 days. Histological analysis of their bones showed the clear presence of GFP positive cells in the Col2.3DeltaTK parabionts, around trabecular bone and on the endosteal and periosteal surfaces. Stromal cell cultures from these Col2.3DeltaTK parabionts did not display mineralized colonies coexpressing GFP. In contrast, scattered GFP positive clusters that contained large cells with morphology similar to osteoclast like cells (OCLs) were observed. These cells were also TRAP positive. They were readily detected in Col2.3DeltaTK mice treated with GCV and transplanted with purified hematopoietic stem cells (HSCs) isolated from pOBCol3.6GFP mice. OCLs were also generated in vitro from osteoclast progenitor cells obtained from pOBCol3.6GFP mice that were defined by the B220- CD3- CD11b- c-fms+ phenotype. Molecular analysis showed that OCLs did not express type I collagen indicating that the Col3.6 promoter contains elements that are active during osteoclastogenesis and are not strictly related to collagen transcription. In summary, we demonstrate that pOBCol3.6 unexpectedly directs the expression of transgenes in the osteoclast lineage and this effect must be considered when utilizing this promoter to study of mesenchymal progenitor cells.