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Radiation-induced alterations of osteogenic and chondrogenic differentiation of human mesenchymal stem cells.

Cruet-Hennequart S, Drougard C, Shaw G, Legendre F, Demoor M, Barry F, Lefaix JL, Galéra P - PLoS ONE (2015)

Bottom Line: Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3.Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis.Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.

View Article: PubMed Central - PubMed

Affiliation: Normandy University, Caen, France; UNICAEN, Laboratoire Microenvironnement Cellulaire et Pathologies (MILPAT), Caen, France; Laboratoire Accueil en Radiobiologie avec les Ions Accélérés (CEA-DSV-IRCM-LARIA), Bd Becquerel, Caen Cedex 5, France; Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland.

ABSTRACT
While human mesenchymal stem cells (hMSCs), either in the bone marrow or in tumour microenvironment could be targeted by radiotherapy, their response is poorly understood. The oxic effects on radiosensitivity, cell cycle progression are largely unknown, and the radiation effects on hMSCs differentiation capacities remained unexplored. Here we analysed hMSCs viability and cell cycle progression in 21% O2 and 3% O2 conditions after medical X-rays irradiation. Differentiation towards osteogenesis and chondrogenesis after irradiation was evaluated through an analysis of differentiation specific genes. Finally, a 3D culture model in hypoxia was used to evaluate chondrogenesis in conditions mimicking the natural hMSCs microenvironment. The hMSCs radiosensitivity was not affected by O2 tension. A decreased number of cells in S phase and an increase in G2/M were observed in both O2 tensions after 16 hours but hMSCs released from the G2/M arrest and proliferated at day 7. Osteogenesis was increased after irradiation with an enhancement of mRNA expression of specific osteogenic genes (alkaline phosphatase, osteopontin). Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3. After induction in monolayers, chondrogenesis was altered after irradiation with an increase in COL1A1 and a decrease in both SOX9 and ACAN mRNA expression. After induction in a 3D culture in hypoxia, chondrogenesis was altered after irradiation with a decrease in COL2A1, ACAN and SOX9 mRNA amounts associated with a RUNX2 increase. Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis. Finally, a radiation-induced impairment of both osteogenesis and chondrogenesis was characterised by a matrix composition alteration, through inhibition of synthesis and/or increased degradation. Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.

No MeSH data available.


Related in: MedlinePlus

Cell cycle progression of hMSCs following X-rays irradiation, under normoxia (21% O2) and physioxia (3% O2) growing conditions.A) Representative histogram, showing the cell cyce phases of hMSCs grown in normoxia for 48h. B-C) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) for 48h before X-rays irradiation, and for the indicated times after irradiation, were harvested and fixed as described in the Material and Methods. DNA was stained with propidium iodide (PI), and DNA content was analyzed by flow cytometry for the determination of cell cycle distribution. Histograms from a representative experiment 16h (A), and 7 days (B) following irradiation are presented. D-E-F) Percentages of cells in the different phases of the cell cycle (as determined using flow cytometry histograms like in A) are presented. Data represent the mean of four independent experiments ± SEM using hMSCs from three different donors for the 16h time point, and up to 9 experiments using hMSCs from five different donors for the 7 day time point. Statistical analysis was performed by unpaired Student t test (* p<0.05, ** p<0.01). G) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) 48h before X-rays irradiation, and for 7 days following irradiation, were fixed and stained using methylene blue. Pictures (Objectives X10) from a representative experiment (n = 4 in normoxia, n = 2 in physioxia) 7 days following irradiation are presented. H) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) 48h before X-rays irradiation, and for 7 days following irradiation, were fixed and stained using the senescence-β Galactosidase kit. Pictures (Objectives X10) from a representative experiment (n = 2) 7 days following irradiation are presented.
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pone.0119334.g002: Cell cycle progression of hMSCs following X-rays irradiation, under normoxia (21% O2) and physioxia (3% O2) growing conditions.A) Representative histogram, showing the cell cyce phases of hMSCs grown in normoxia for 48h. B-C) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) for 48h before X-rays irradiation, and for the indicated times after irradiation, were harvested and fixed as described in the Material and Methods. DNA was stained with propidium iodide (PI), and DNA content was analyzed by flow cytometry for the determination of cell cycle distribution. Histograms from a representative experiment 16h (A), and 7 days (B) following irradiation are presented. D-E-F) Percentages of cells in the different phases of the cell cycle (as determined using flow cytometry histograms like in A) are presented. Data represent the mean of four independent experiments ± SEM using hMSCs from three different donors for the 16h time point, and up to 9 experiments using hMSCs from five different donors for the 7 day time point. Statistical analysis was performed by unpaired Student t test (* p<0.05, ** p<0.01). G) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) 48h before X-rays irradiation, and for 7 days following irradiation, were fixed and stained using methylene blue. Pictures (Objectives X10) from a representative experiment (n = 4 in normoxia, n = 2 in physioxia) 7 days following irradiation are presented. H) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) 48h before X-rays irradiation, and for 7 days following irradiation, were fixed and stained using the senescence-β Galactosidase kit. Pictures (Objectives X10) from a representative experiment (n = 2) 7 days following irradiation are presented.

Mentions: Cells were seeded and grown either in normoxia or in physioxia 48h before irradiation. Detailed cell cycle analysis was performed at the time of irradiation (T0-48h culture), and both shortly (16h) and late (7 days) after irradiation. At the indicated times following irradiation, cell cycle progression was analysed by flow cytometry using a Beckman Coulter Gallios flow cytometer (Federative Research Structure ICORE platform, University of Caen/Lower-Normandy-France). Briefly, trypsinised cells were fixed in 70% ethanol and stored at -20°C until staining, for 30 min at 37°C with a PBS solution containing 20 μg/ml propidium iodide and 100 μg/ml RNAse. Data was analysed using Kaluza software. Linear gates have been used to define each subpopulation (G0/G1, S and G2/M), as presented in Fig 2. The subG1 population (representative of the apoptotic population, (less than 5%), and any post G2/M subpopulation (representative of endoreplication, less than 5%), have been gated out, in order to get solely the percentages of the cycling population in each subpopulation (G0/G1, S and G2/M).


Radiation-induced alterations of osteogenic and chondrogenic differentiation of human mesenchymal stem cells.

Cruet-Hennequart S, Drougard C, Shaw G, Legendre F, Demoor M, Barry F, Lefaix JL, Galéra P - PLoS ONE (2015)

Cell cycle progression of hMSCs following X-rays irradiation, under normoxia (21% O2) and physioxia (3% O2) growing conditions.A) Representative histogram, showing the cell cyce phases of hMSCs grown in normoxia for 48h. B-C) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) for 48h before X-rays irradiation, and for the indicated times after irradiation, were harvested and fixed as described in the Material and Methods. DNA was stained with propidium iodide (PI), and DNA content was analyzed by flow cytometry for the determination of cell cycle distribution. Histograms from a representative experiment 16h (A), and 7 days (B) following irradiation are presented. D-E-F) Percentages of cells in the different phases of the cell cycle (as determined using flow cytometry histograms like in A) are presented. Data represent the mean of four independent experiments ± SEM using hMSCs from three different donors for the 16h time point, and up to 9 experiments using hMSCs from five different donors for the 7 day time point. Statistical analysis was performed by unpaired Student t test (* p<0.05, ** p<0.01). G) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) 48h before X-rays irradiation, and for 7 days following irradiation, were fixed and stained using methylene blue. Pictures (Objectives X10) from a representative experiment (n = 4 in normoxia, n = 2 in physioxia) 7 days following irradiation are presented. H) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) 48h before X-rays irradiation, and for 7 days following irradiation, were fixed and stained using the senescence-β Galactosidase kit. Pictures (Objectives X10) from a representative experiment (n = 2) 7 days following irradiation are presented.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383487&req=5

pone.0119334.g002: Cell cycle progression of hMSCs following X-rays irradiation, under normoxia (21% O2) and physioxia (3% O2) growing conditions.A) Representative histogram, showing the cell cyce phases of hMSCs grown in normoxia for 48h. B-C) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) for 48h before X-rays irradiation, and for the indicated times after irradiation, were harvested and fixed as described in the Material and Methods. DNA was stained with propidium iodide (PI), and DNA content was analyzed by flow cytometry for the determination of cell cycle distribution. Histograms from a representative experiment 16h (A), and 7 days (B) following irradiation are presented. D-E-F) Percentages of cells in the different phases of the cell cycle (as determined using flow cytometry histograms like in A) are presented. Data represent the mean of four independent experiments ± SEM using hMSCs from three different donors for the 16h time point, and up to 9 experiments using hMSCs from five different donors for the 7 day time point. Statistical analysis was performed by unpaired Student t test (* p<0.05, ** p<0.01). G) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) 48h before X-rays irradiation, and for 7 days following irradiation, were fixed and stained using methylene blue. Pictures (Objectives X10) from a representative experiment (n = 4 in normoxia, n = 2 in physioxia) 7 days following irradiation are presented. H) hMSCs grown in normoxia (21% O2) or physioxia (3% O2) 48h before X-rays irradiation, and for 7 days following irradiation, were fixed and stained using the senescence-β Galactosidase kit. Pictures (Objectives X10) from a representative experiment (n = 2) 7 days following irradiation are presented.
Mentions: Cells were seeded and grown either in normoxia or in physioxia 48h before irradiation. Detailed cell cycle analysis was performed at the time of irradiation (T0-48h culture), and both shortly (16h) and late (7 days) after irradiation. At the indicated times following irradiation, cell cycle progression was analysed by flow cytometry using a Beckman Coulter Gallios flow cytometer (Federative Research Structure ICORE platform, University of Caen/Lower-Normandy-France). Briefly, trypsinised cells were fixed in 70% ethanol and stored at -20°C until staining, for 30 min at 37°C with a PBS solution containing 20 μg/ml propidium iodide and 100 μg/ml RNAse. Data was analysed using Kaluza software. Linear gates have been used to define each subpopulation (G0/G1, S and G2/M), as presented in Fig 2. The subG1 population (representative of the apoptotic population, (less than 5%), and any post G2/M subpopulation (representative of endoreplication, less than 5%), have been gated out, in order to get solely the percentages of the cycling population in each subpopulation (G0/G1, S and G2/M).

Bottom Line: Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3.Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis.Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.

View Article: PubMed Central - PubMed

Affiliation: Normandy University, Caen, France; UNICAEN, Laboratoire Microenvironnement Cellulaire et Pathologies (MILPAT), Caen, France; Laboratoire Accueil en Radiobiologie avec les Ions Accélérés (CEA-DSV-IRCM-LARIA), Bd Becquerel, Caen Cedex 5, France; Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland.

ABSTRACT
While human mesenchymal stem cells (hMSCs), either in the bone marrow or in tumour microenvironment could be targeted by radiotherapy, their response is poorly understood. The oxic effects on radiosensitivity, cell cycle progression are largely unknown, and the radiation effects on hMSCs differentiation capacities remained unexplored. Here we analysed hMSCs viability and cell cycle progression in 21% O2 and 3% O2 conditions after medical X-rays irradiation. Differentiation towards osteogenesis and chondrogenesis after irradiation was evaluated through an analysis of differentiation specific genes. Finally, a 3D culture model in hypoxia was used to evaluate chondrogenesis in conditions mimicking the natural hMSCs microenvironment. The hMSCs radiosensitivity was not affected by O2 tension. A decreased number of cells in S phase and an increase in G2/M were observed in both O2 tensions after 16 hours but hMSCs released from the G2/M arrest and proliferated at day 7. Osteogenesis was increased after irradiation with an enhancement of mRNA expression of specific osteogenic genes (alkaline phosphatase, osteopontin). Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3. After induction in monolayers, chondrogenesis was altered after irradiation with an increase in COL1A1 and a decrease in both SOX9 and ACAN mRNA expression. After induction in a 3D culture in hypoxia, chondrogenesis was altered after irradiation with a decrease in COL2A1, ACAN and SOX9 mRNA amounts associated with a RUNX2 increase. Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis. Finally, a radiation-induced impairment of both osteogenesis and chondrogenesis was characterised by a matrix composition alteration, through inhibition of synthesis and/or increased degradation. Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.

No MeSH data available.


Related in: MedlinePlus