Background: Special attention is being given to the use of adult stem cells in tissue repair; in particular the mesenchymal stem cells (MSCs) have been studied extensively because of their multipotentiality, and because of their ability to self-renew, to respond to different factors and to give rise to various differentiated cell types, including chondrocytes and osteocytes. Parathyroid hormone (PTH(1-34)) is a key molecule regulating calcium and phosphate homeostasis. Recently it has been demonstrated relevant direct effects of PTH on the hematopoietic system in a mouse model. In this model, it has been observed an increase in the number of stem cells after PTH injection. The present study was designed to test the effects of PTH and/or hypoxia on MSC growth in vitro. Methods: MSCs were extracted from femurs of rats and maintained in culture with alpha-MEM in the presence of 10% FBS serum for three passages (P3), then the MSCs were subjected to the following experimental protocols: A) Cell growth in normoxia: 1) alpha-MEM medium alone (control): MSC cells were seeded at a density of 8-10 x 104 cells/ml and placed in 5% CO2 incubator for 12 days; 2) alpha-MEM+PTH (PTH treated): the same procedure was repeated in the presence of PTH(1-34) at the concentration of 100nM for 12 days. Cell viability was assessed by MTT test at 3, 6, 9 and 12 days of colture. B) Cell growth in hypoxia: 3) MSCs were exposed to PTH(1-34) 100 nM concentration for 24h (PTH pre-treatment); after PTH removal the cells were incubated for 3 days in hypoxic chamber (O2 3%); cell viability was assessed by MTT test and compared with MSC incubated for 3 days in hypoxic chamber without PTH pre-treatment. 4) For comparative purpose, cell viability was also assed in MSC incubated for 3 days only in normoxic conditions, with and without PTH pre-treatment. Results: A) Cell growth in normoxia: treatment with PTH significantly enhanced cell growth compared to the control group for the entire duration of treatment. The effect of PTH on cell growth was already appreciable after 3 days of treatment and increased with time. B) Cell growth in hypoxia: pre-treatment with PTH significantly improved cell viability/survival in hypoxia (i.e., limited hypoxia-induced cell death). Moreover, pre-treatment with PTH was sufficient to increase cell growth in normoxic conditions. Conclusions: our preliminary results demonstrate an important pro-growth (anti-death) effect of PTH on MSC. Importantly, the significant protection exerted by PTH(1-34) against hypoxia-induced cell death suggests that PTH-MSC interaction plays an important role in bone repair, even in the presence of tissue hypoperfusion as such occurring in bone fractures.
PTH Enhances Mesenchymal Stem Cell (MSC) Growth and Reduces hypoxia-induced MSC death
PERRELLI, MARIA-GIULIA;PENNA, Claudia;PAGLIARO, Pasquale;PORTIGLIATTI BARBOS, Michele;MOGNETTI, Barbara
2011-01-01
Abstract
Background: Special attention is being given to the use of adult stem cells in tissue repair; in particular the mesenchymal stem cells (MSCs) have been studied extensively because of their multipotentiality, and because of their ability to self-renew, to respond to different factors and to give rise to various differentiated cell types, including chondrocytes and osteocytes. Parathyroid hormone (PTH(1-34)) is a key molecule regulating calcium and phosphate homeostasis. Recently it has been demonstrated relevant direct effects of PTH on the hematopoietic system in a mouse model. In this model, it has been observed an increase in the number of stem cells after PTH injection. The present study was designed to test the effects of PTH and/or hypoxia on MSC growth in vitro. Methods: MSCs were extracted from femurs of rats and maintained in culture with alpha-MEM in the presence of 10% FBS serum for three passages (P3), then the MSCs were subjected to the following experimental protocols: A) Cell growth in normoxia: 1) alpha-MEM medium alone (control): MSC cells were seeded at a density of 8-10 x 104 cells/ml and placed in 5% CO2 incubator for 12 days; 2) alpha-MEM+PTH (PTH treated): the same procedure was repeated in the presence of PTH(1-34) at the concentration of 100nM for 12 days. Cell viability was assessed by MTT test at 3, 6, 9 and 12 days of colture. B) Cell growth in hypoxia: 3) MSCs were exposed to PTH(1-34) 100 nM concentration for 24h (PTH pre-treatment); after PTH removal the cells were incubated for 3 days in hypoxic chamber (O2 3%); cell viability was assessed by MTT test and compared with MSC incubated for 3 days in hypoxic chamber without PTH pre-treatment. 4) For comparative purpose, cell viability was also assed in MSC incubated for 3 days only in normoxic conditions, with and without PTH pre-treatment. Results: A) Cell growth in normoxia: treatment with PTH significantly enhanced cell growth compared to the control group for the entire duration of treatment. The effect of PTH on cell growth was already appreciable after 3 days of treatment and increased with time. B) Cell growth in hypoxia: pre-treatment with PTH significantly improved cell viability/survival in hypoxia (i.e., limited hypoxia-induced cell death). Moreover, pre-treatment with PTH was sufficient to increase cell growth in normoxic conditions. Conclusions: our preliminary results demonstrate an important pro-growth (anti-death) effect of PTH on MSC. Importantly, the significant protection exerted by PTH(1-34) against hypoxia-induced cell death suggests that PTH-MSC interaction plays an important role in bone repair, even in the presence of tissue hypoperfusion as such occurring in bone fractures.
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