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Developmental restriction of embryonic mesen chymal stem cells as characterized by their in vitro potential for differentiation
398
Abstracts
Bone Vol. 16, No. 3 March 1995:391-410
21 INFLUENCE OF TAMOXIFEN ON HUMAN OSTEOBLASTS IN VITRO. G. Leb, R. Wildburaer. M Schmid, T. Department of Medicine, University of
22 PROLIFERATION OF K. Weber. S. Kralievic. Lauermann, G.J. Kreis, Graz, Austria
Introduction: Tamoxifen, a synthetic antiestrogen, is the treatment of choice for women suffering from hormonally responsive breast cancer. Clinical studies give evidence for an unexpected increase of bone mineral density, at least at the vertebral site, during tamoxifen treatment (NEJM 1992; 326:852). In the present study we investigated the effect of tamoxifen on proliferation of human osteoblasts in vitro. Methods: Human osteoblasts were isolated from small fragments of female trabecular bone by mechanical and enzymatic technics. Tamoxifen (0.001-10 uM) was added to cultured cells in 96-well plates for 48 hours. Growth stimulation was measured by Ho3-thymidine incorporation. Results are shown as counts per minute, each mean value represents 6 experiments. Results: mean sd % ~ control t - ted control 229 41 100% tamoxifen 0.001 366 60 160% p<0.001 tamoxifen 0.01 438 112 191% p<0.01 tamoxifen 0.1 359 121 157% p<0.05 tamoxifen 1 317 71 138% p<0.05 tamoxifen 10 35 11 toxic effect C o n c l u s i o n s : In spite of its antiestrogenic properties tamoxifen stimulates proliferation of human osteoblasts in vitro in a concentration-dependent manner with a maximum at 0.01 uM. Concentrations higher than 1 uM showed the expected toxic effect. These findings might explain the clinical observed increase of bone mineral density in women treated with tamoxifen.
DEVELOPMENTAL RESTRICTION OF EMBRYONIC MESEN. CHYMAL STEM CELLS AS CHARACTERIZED BY THEIR Ih VITRO POTENTIAL FOR DIFFERENTIATION. Cyril D. Toma Jonathan L. Schaffer and Louis C. Gerstenfeld. Laborator~ for the Study of Skeletal Disorders and Rehabilitation Children's Hospital; Department of Orthopedic Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. The development of skeletal tissues is dependent upon the proliferation and differentiation of skeletally derived progenitor cells. In embryonic calvaria, bone development occurs via the intramembranous pathway whereby mesenchymal stem cells differentiate directly into osteoblasts. Typically, the cartilage phenotype is not observed in cultures of embryonic calvarial cells. This study focused on the potential of calvarial stem cell populations isolated from embryos of differing ages to differentiate into specific cell types of the mesenchymal lineage as a function of their in vivo developmental stage. To assess the potential of these cells to differentiate into their multiple lineages, the expression of collagen types I, II and X and several bone specific mRNA and proteins and the histochemical distribution of lineage specific markers for osteoblasts and chondrocytes were examined. Depending on culture conditions, cells isolated from 12 day embryonic calvaria were capable of assuming either the osteoblastic or chondrocytic lineage progression pathway. In contrast, 17 day embryonic cell populations had a restricted developmental potential and expressed only the osteoblastic phenotype. In conclusion, an inherent potential of the undifferentiated cells within the calvarial cell population to progress to either chondrocytes or osteoblasts was observed. The restriction of the phenotypic potential of these cells is due to the in vivo milieu. Thus, during tissue development in vivo, the surrounding environmental conditions are regulated such that there is a progressive decrease of the stem cell population and there is a loss of the cellular responsiveness to extrinsic modulators.
23 OSTEOBLAST PHENOTYPIC EXPRESSION AFTER MECHANICAL PERTURBATION WITH A DYNAMIC, ISOTROPIC, SIAXIALLY UNIFORM STRAIN. CD Toma. LC Gerstenfeld. ML Gray and JL Schaffer. Lab for the Study of Skeletal Disorders and Rehabilitation, Children's Hospital, Harvard-MIT Health Science and Technology, Department of Orthopedic Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. A basic tenant of skeletal tissue biology is that mechanical factors affect bone's structural development and molecular composition. The objective of this investigation was to elucidate the molecular responses of osteoblasts (OB) to mechanical perturbations using a well characterized OB culture system which temporally progresses in vitro through three distinct phases of development (proliferation, differentiative phenotypic expression and extracellular matrix mineralization) in conjunction with a device for mechanically deforming a flexible cellladen substrate that provides independent control of the spatial and temporal biaxial strain distribution imposed on the membrane. Embryonic 12d chicken OB cells were cultured on a surface treated flexible elastomeric surface. During the differentiative phase of OB development, cells were mechanically perturbed at 1-2% uniform biaxial strain at 0.25 Hz with an 8% duty cycle (2 hrs/day) for 4 days (3 experiments at each strain, n_>6for each group). Cells were collected and analyzed for APase activity, DNA content, collagen, protein, calcium and osteopontin (OPN) accumulation and collagen type 1, osteocalcin and OPN steady state mRNA. There were statistically significant decreases in APase activity and OPN accumulation relative to static controls. No statistically significant differences were observed for the DNA content, collagen, protein or calcium accumulation. Northern blot analysis demonstrated gene level responses that were consistent with the protein level responses. Expression of an osteocyte stage specific surface marker normalized to total cell nuclei was increased. In conclusion, we speculate that the decreases in membrane bound alkaline phosphatase activity and attachment associated OPN reflect membrane and attachment site alterations with mechanical perturbation. When the data are considered in the context of the normal temporal progression for the controls it appears that mechanical perturbation may increase the rate of developmental progression.
24 ROLE OF PARATHYROID HORMONE (PTH) IN OSTEOCHONDROGENIC DEVELOPMENT OF BONE MORPHOGENETIC PROTEIN ( B M P ) - TRANSFECTED MESENCHYMAL PROGENITORS (C3H10TIA) A. Hollnag¢l, M, Ahr¢ns, D, $~hr6der, H. Mayer. G. Gross. Bone Research Group, Gesellschaft fiir Biotechnologische Forschung (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany Permanent expression of genes encoding human BMPs in mesenchymal progenitor cells (C3H10TI/~) induce differentiation into three distinct cell lineages: adipogenic, ehondrogenic and osteogenic lineages (1). We further investigated as to whether or not parathyroid hormone (PTH) in BMP-transfected C3H10TV= cells stimulates the differentiation into the osteogenic and/or chondrogenic lineage since it is known that in rive longtime application of PTH leads to increased bone growth. Indeed, PTH(1-34) application leads to increased rates of differentiation into the ostcoblastic and chondroblastic lineage in B M P - t r a n s f e c t e d C3H10TI/z cells which was further substantiated by coexpressing the recombinant rat-PTH-receptor in these cells. The action of PTH upon osteochondrogenic development takes place during the early commitment period of these lineages, is mediated by the aminoterminal PTH fragment (1-34) and is transferred by the cAMP signalling cascade. However, application of PTH late in osteochondrogenic development inhibits terminal differentiation. Conclusion: The positive and negative influence of PTH upon osteochondrogenic development is dependent on the developmental state of the progenitors. This observation may reconcile many controversial studies on differentiating or dedifferentiating effects of PTH. THE
1. Ahrens, M., Ankenbauer, T., Schr6der, D., Hollnagel, A., Mayer, H., and Gross, G. (1993) DNA and Cell Biology 12:870-871
Abstracts
Bone Vol. 16, No. 3 March 1995:391-410
21 INFLUENCE OF TAMOXIFEN ON HUMAN OSTEOBLASTS IN VITRO. G. Leb, R. Wildburaer. M Schmid, T. Department of Medicine, University of
22 PROLIFERATION OF K. Weber. S. Kralievic. Lauermann, G.J. Kreis, Graz, Austria
Introduction: Tamoxifen, a synthetic antiestrogen, is the treatment of choice for women suffering from hormonally responsive breast cancer. Clinical studies give evidence for an unexpected increase of bone mineral density, at least at the vertebral site, during tamoxifen treatment (NEJM 1992; 326:852). In the present study we investigated the effect of tamoxifen on proliferation of human osteoblasts in vitro. Methods: Human osteoblasts were isolated from small fragments of female trabecular bone by mechanical and enzymatic technics. Tamoxifen (0.001-10 uM) was added to cultured cells in 96-well plates for 48 hours. Growth stimulation was measured by Ho3-thymidine incorporation. Results are shown as counts per minute, each mean value represents 6 experiments. Results: mean sd % ~ control t - ted control 229 41 100% tamoxifen 0.001 366 60 160% p<0.001 tamoxifen 0.01 438 112 191% p<0.01 tamoxifen 0.1 359 121 157% p<0.05 tamoxifen 1 317 71 138% p<0.05 tamoxifen 10 35 11 toxic effect C o n c l u s i o n s : In spite of its antiestrogenic properties tamoxifen stimulates proliferation of human osteoblasts in vitro in a concentration-dependent manner with a maximum at 0.01 uM. Concentrations higher than 1 uM showed the expected toxic effect. These findings might explain the clinical observed increase of bone mineral density in women treated with tamoxifen.
DEVELOPMENTAL RESTRICTION OF EMBRYONIC MESEN. CHYMAL STEM CELLS AS CHARACTERIZED BY THEIR Ih VITRO POTENTIAL FOR DIFFERENTIATION. Cyril D. Toma Jonathan L. Schaffer and Louis C. Gerstenfeld. Laborator~ for the Study of Skeletal Disorders and Rehabilitation Children's Hospital; Department of Orthopedic Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. The development of skeletal tissues is dependent upon the proliferation and differentiation of skeletally derived progenitor cells. In embryonic calvaria, bone development occurs via the intramembranous pathway whereby mesenchymal stem cells differentiate directly into osteoblasts. Typically, the cartilage phenotype is not observed in cultures of embryonic calvarial cells. This study focused on the potential of calvarial stem cell populations isolated from embryos of differing ages to differentiate into specific cell types of the mesenchymal lineage as a function of their in vivo developmental stage. To assess the potential of these cells to differentiate into their multiple lineages, the expression of collagen types I, II and X and several bone specific mRNA and proteins and the histochemical distribution of lineage specific markers for osteoblasts and chondrocytes were examined. Depending on culture conditions, cells isolated from 12 day embryonic calvaria were capable of assuming either the osteoblastic or chondrocytic lineage progression pathway. In contrast, 17 day embryonic cell populations had a restricted developmental potential and expressed only the osteoblastic phenotype. In conclusion, an inherent potential of the undifferentiated cells within the calvarial cell population to progress to either chondrocytes or osteoblasts was observed. The restriction of the phenotypic potential of these cells is due to the in vivo milieu. Thus, during tissue development in vivo, the surrounding environmental conditions are regulated such that there is a progressive decrease of the stem cell population and there is a loss of the cellular responsiveness to extrinsic modulators.
23 OSTEOBLAST PHENOTYPIC EXPRESSION AFTER MECHANICAL PERTURBATION WITH A DYNAMIC, ISOTROPIC, SIAXIALLY UNIFORM STRAIN. CD Toma. LC Gerstenfeld. ML Gray and JL Schaffer. Lab for the Study of Skeletal Disorders and Rehabilitation, Children's Hospital, Harvard-MIT Health Science and Technology, Department of Orthopedic Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. A basic tenant of skeletal tissue biology is that mechanical factors affect bone's structural development and molecular composition. The objective of this investigation was to elucidate the molecular responses of osteoblasts (OB) to mechanical perturbations using a well characterized OB culture system which temporally progresses in vitro through three distinct phases of development (proliferation, differentiative phenotypic expression and extracellular matrix mineralization) in conjunction with a device for mechanically deforming a flexible cellladen substrate that provides independent control of the spatial and temporal biaxial strain distribution imposed on the membrane. Embryonic 12d chicken OB cells were cultured on a surface treated flexible elastomeric surface. During the differentiative phase of OB development, cells were mechanically perturbed at 1-2% uniform biaxial strain at 0.25 Hz with an 8% duty cycle (2 hrs/day) for 4 days (3 experiments at each strain, n_>6for each group). Cells were collected and analyzed for APase activity, DNA content, collagen, protein, calcium and osteopontin (OPN) accumulation and collagen type 1, osteocalcin and OPN steady state mRNA. There were statistically significant decreases in APase activity and OPN accumulation relative to static controls. No statistically significant differences were observed for the DNA content, collagen, protein or calcium accumulation. Northern blot analysis demonstrated gene level responses that were consistent with the protein level responses. Expression of an osteocyte stage specific surface marker normalized to total cell nuclei was increased. In conclusion, we speculate that the decreases in membrane bound alkaline phosphatase activity and attachment associated OPN reflect membrane and attachment site alterations with mechanical perturbation. When the data are considered in the context of the normal temporal progression for the controls it appears that mechanical perturbation may increase the rate of developmental progression.
24 ROLE OF PARATHYROID HORMONE (PTH) IN OSTEOCHONDROGENIC DEVELOPMENT OF BONE MORPHOGENETIC PROTEIN ( B M P ) - TRANSFECTED MESENCHYMAL PROGENITORS (C3H10TIA) A. Hollnag¢l, M, Ahr¢ns, D, $~hr6der, H. Mayer. G. Gross. Bone Research Group, Gesellschaft fiir Biotechnologische Forschung (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany Permanent expression of genes encoding human BMPs in mesenchymal progenitor cells (C3H10TI/~) induce differentiation into three distinct cell lineages: adipogenic, ehondrogenic and osteogenic lineages (1). We further investigated as to whether or not parathyroid hormone (PTH) in BMP-transfected C3H10TV= cells stimulates the differentiation into the osteogenic and/or chondrogenic lineage since it is known that in rive longtime application of PTH leads to increased bone growth. Indeed, PTH(1-34) application leads to increased rates of differentiation into the ostcoblastic and chondroblastic lineage in B M P - t r a n s f e c t e d C3H10TI/z cells which was further substantiated by coexpressing the recombinant rat-PTH-receptor in these cells. The action of PTH upon osteochondrogenic development takes place during the early commitment period of these lineages, is mediated by the aminoterminal PTH fragment (1-34) and is transferred by the cAMP signalling cascade. However, application of PTH late in osteochondrogenic development inhibits terminal differentiation. Conclusion: The positive and negative influence of PTH upon osteochondrogenic development is dependent on the developmental state of the progenitors. This observation may reconcile many controversial studies on differentiating or dedifferentiating effects of PTH. THE
1. Ahrens, M., Ankenbauer, T., Schr6der, D., Hollnagel, A., Mayer, H., and Gross, G. (1993) DNA and Cell Biology 12:870-871