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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

Effect of X-irradiation on osteogenic differentiation of hMSCs.hMSCs were mock-irradiated or irradiated at 6 Gy and 10 Gy. 16h following X-rays irradiation, osteogenic differentiation (OS-hMSCs) was induced and maintained using osteogenic differentiation medium for 21 days as described in Material and Methods. Undifferentiated hMSCs (ND-hMSCs) were grown in regular culture medium for 21 days. A) Light microscopy photographs of hMSCs grown for 21 days in complete regular culture media (ND-hMSCs) or osteogenic differentiation media (OS-hMSCs). Photographs are representative of data from (Objectives X10) 3 donors. B) Photographs of hMSCs after methylene blue and alizarin red S staining, 21 days following osteogenic differentiation after irradiation (0-6-10 Gy). Methylene blue staining was used to assess hMSCs morphology in undifferentiated (ND-hMSCs) and osteogenically-differentiated cells (OS-hMSCs) after irradiation. Alizarin red S staining was used to detect the presence of calcium deposition in undifferentiated (ND-hMSCs) and osteogenically- differentiated cells (OS-hMSCs) after X-rays irradiation. Representative photographs (Objectives X10) of 3 donors. C) Real-time RT-PCR analysis of relative mRNA expression of the indicated genes (ALP-alkaline phosphatase, OPN-osteopontin, BSP-Bone sialoprotein, SOX9-Sox9 transcription factor, OCN-Osteocalcin, RUNX2-runt-related transcription factor 2, COL1A1-type I collagen, COL10A1-type X collagen, COL2A1-type II collagen) in undifferentiated hMSCs (ND-hMSCs) and osteogenically differentiated hMSCs (OS-hMSCs) after irradiation (0-6-10 Gy). Gene expression is normalized against the endogenous reference gene, RPL13A. Results are expressed as relative mRNA expression as compared to the control condition (undifferentiated hMSCs, 0 Gy condition) set at the arbitrary unit of 1 Box and whiskers plots (Min to Max. Bar at median and + at mean) of data derived from 5 donors. Statistical analysis (One-Way ANOVA, with Tukey post-test) was performed and statistical significance is shown with, * for p<0.05, and ** for p< 0.01. D) Western blot analysis of type II collagen, type I collagen, and GAPDH in undifferentiated (ND-hMSCs) and osteogenically differentiated cells (OS-hMSCs) after irradiation. E) Real-time RT-PCR analysis of expression levels of the indicated genes (MMP-1, MMP-3, and MMP-13) in undifferentiated hMSCs (ND-hMSCs) and osteogenically differentiated hMSCs (CH 3D-hMSCs) after irradiation (0-6-10 Gy). Gene expression is normalized against the endogenous reference gene, RPL13A. Results are expressed as relative mRNA expression as compared to the control condition (undifferentiated hMSCs, 0 Gy condition) set at the arbitrary unit of 1. Box and whiskers (Min to Max. Bar at median and + at mean) of 4 independent experiments. Statistical analysis (One-Way ANOVA, with Tukey post-test) were performed and statistical significance is shown with, * for p<0.05, and ** for p< 0.01.
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pone.0119334.g003: Effect of X-irradiation on osteogenic differentiation of hMSCs.hMSCs were mock-irradiated or irradiated at 6 Gy and 10 Gy. 16h following X-rays irradiation, osteogenic differentiation (OS-hMSCs) was induced and maintained using osteogenic differentiation medium for 21 days as described in Material and Methods. Undifferentiated hMSCs (ND-hMSCs) were grown in regular culture medium for 21 days. A) Light microscopy photographs of hMSCs grown for 21 days in complete regular culture media (ND-hMSCs) or osteogenic differentiation media (OS-hMSCs). Photographs are representative of data from (Objectives X10) 3 donors. B) Photographs of hMSCs after methylene blue and alizarin red S staining, 21 days following osteogenic differentiation after irradiation (0-6-10 Gy). Methylene blue staining was used to assess hMSCs morphology in undifferentiated (ND-hMSCs) and osteogenically-differentiated cells (OS-hMSCs) after irradiation. Alizarin red S staining was used to detect the presence of calcium deposition in undifferentiated (ND-hMSCs) and osteogenically- differentiated cells (OS-hMSCs) after X-rays irradiation. Representative photographs (Objectives X10) of 3 donors. C) Real-time RT-PCR analysis of relative mRNA expression of the indicated genes (ALP-alkaline phosphatase, OPN-osteopontin, BSP-Bone sialoprotein, SOX9-Sox9 transcription factor, OCN-Osteocalcin, RUNX2-runt-related transcription factor 2, COL1A1-type I collagen, COL10A1-type X collagen, COL2A1-type II collagen) in undifferentiated hMSCs (ND-hMSCs) and osteogenically differentiated hMSCs (OS-hMSCs) after irradiation (0-6-10 Gy). Gene expression is normalized against the endogenous reference gene, RPL13A. Results are expressed as relative mRNA expression as compared to the control condition (undifferentiated hMSCs, 0 Gy condition) set at the arbitrary unit of 1 Box and whiskers plots (Min to Max. Bar at median and + at mean) of data derived from 5 donors. Statistical analysis (One-Way ANOVA, with Tukey post-test) was performed and statistical significance is shown with, * for p<0.05, and ** for p< 0.01. D) Western blot analysis of type II collagen, type I collagen, and GAPDH in undifferentiated (ND-hMSCs) and osteogenically differentiated cells (OS-hMSCs) after irradiation. E) Real-time RT-PCR analysis of expression levels of the indicated genes (MMP-1, MMP-3, and MMP-13) in undifferentiated hMSCs (ND-hMSCs) and osteogenically differentiated hMSCs (CH 3D-hMSCs) after irradiation (0-6-10 Gy). Gene expression is normalized against the endogenous reference gene, RPL13A. Results are expressed as relative mRNA expression as compared to the control condition (undifferentiated hMSCs, 0 Gy condition) set at the arbitrary unit of 1. Box and whiskers (Min to Max. Bar at median and + at mean) of 4 independent experiments. Statistical analysis (One-Way ANOVA, with Tukey post-test) were performed and statistical significance is shown with, * for p<0.05, and ** for p< 0.01.

Mentions: Osteogenic differentiation was induced after X-irradiation and assessed 21 days later. As seen in light microscopy photographs (Fig 3A), dense, refractile deposits, indicative of mineralization were seen in osteogenically differentiated hMSCs but not in the undifferentiated hMSCs (compare ND-hMSCs and OS-hMSCs at 0 Gy). In addition, a dose-dependent increase in the density of these deposits was observed in the irradiated and osteogenically differentiated hMSCs (OS-hMSCs), with denser deposits being observed after 10 Gy compared to 6 Gy. In order to specifically assess that these deposits were genuine calcium deposits, we performed an Alizarin red solution staining. Methylene blue staining was also performed in order to show the morphological changes associated with osteoblastic differentiation. As seen in Fig 3B (methylene blue), osteogenically differentiated hMSCs (OS-hMSCs) presented a more cuboidal morphology, characteristic of osteoblasts than the undifferentiated hMSCs, indicating proper differentiation induction. Moreover, no alizarin red staining was observed in the undifferentiated hMSCs, while osteogenically differentiated hMSCs exhibited a strong red staining indicative of calcium deposition and osteogenic differentiation. hMSCs, that had been osteogenically differentiated after 6 Gy and 10 Gy, despite showing a lower density than the undifferentiated hMSCS, presented a more pronounced alizarin red staining compared to the mock-irradiated OS-hMSCs, and displayed mineralized nodules, indicative of increased differentiation after 6 and 10 Gy.


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)

Effect of X-irradiation on osteogenic differentiation of hMSCs.hMSCs were mock-irradiated or irradiated at 6 Gy and 10 Gy. 16h following X-rays irradiation, osteogenic differentiation (OS-hMSCs) was induced and maintained using osteogenic differentiation medium for 21 days as described in Material and Methods. Undifferentiated hMSCs (ND-hMSCs) were grown in regular culture medium for 21 days. A) Light microscopy photographs of hMSCs grown for 21 days in complete regular culture media (ND-hMSCs) or osteogenic differentiation media (OS-hMSCs). Photographs are representative of data from (Objectives X10) 3 donors. B) Photographs of hMSCs after methylene blue and alizarin red S staining, 21 days following osteogenic differentiation after irradiation (0-6-10 Gy). Methylene blue staining was used to assess hMSCs morphology in undifferentiated (ND-hMSCs) and osteogenically-differentiated cells (OS-hMSCs) after irradiation. Alizarin red S staining was used to detect the presence of calcium deposition in undifferentiated (ND-hMSCs) and osteogenically- differentiated cells (OS-hMSCs) after X-rays irradiation. Representative photographs (Objectives X10) of 3 donors. C) Real-time RT-PCR analysis of relative mRNA expression of the indicated genes (ALP-alkaline phosphatase, OPN-osteopontin, BSP-Bone sialoprotein, SOX9-Sox9 transcription factor, OCN-Osteocalcin, RUNX2-runt-related transcription factor 2, COL1A1-type I collagen, COL10A1-type X collagen, COL2A1-type II collagen) in undifferentiated hMSCs (ND-hMSCs) and osteogenically differentiated hMSCs (OS-hMSCs) after irradiation (0-6-10 Gy). Gene expression is normalized against the endogenous reference gene, RPL13A. Results are expressed as relative mRNA expression as compared to the control condition (undifferentiated hMSCs, 0 Gy condition) set at the arbitrary unit of 1 Box and whiskers plots (Min to Max. Bar at median and + at mean) of data derived from 5 donors. Statistical analysis (One-Way ANOVA, with Tukey post-test) was performed and statistical significance is shown with, * for p<0.05, and ** for p< 0.01. D) Western blot analysis of type II collagen, type I collagen, and GAPDH in undifferentiated (ND-hMSCs) and osteogenically differentiated cells (OS-hMSCs) after irradiation. E) Real-time RT-PCR analysis of expression levels of the indicated genes (MMP-1, MMP-3, and MMP-13) in undifferentiated hMSCs (ND-hMSCs) and osteogenically differentiated hMSCs (CH 3D-hMSCs) after irradiation (0-6-10 Gy). Gene expression is normalized against the endogenous reference gene, RPL13A. Results are expressed as relative mRNA expression as compared to the control condition (undifferentiated hMSCs, 0 Gy condition) set at the arbitrary unit of 1. Box and whiskers (Min to Max. Bar at median and + at mean) of 4 independent experiments. Statistical analysis (One-Way ANOVA, with Tukey post-test) were performed and statistical significance is shown with, * for p<0.05, and ** for p< 0.01.
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Show All Figures
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pone.0119334.g003: Effect of X-irradiation on osteogenic differentiation of hMSCs.hMSCs were mock-irradiated or irradiated at 6 Gy and 10 Gy. 16h following X-rays irradiation, osteogenic differentiation (OS-hMSCs) was induced and maintained using osteogenic differentiation medium for 21 days as described in Material and Methods. Undifferentiated hMSCs (ND-hMSCs) were grown in regular culture medium for 21 days. A) Light microscopy photographs of hMSCs grown for 21 days in complete regular culture media (ND-hMSCs) or osteogenic differentiation media (OS-hMSCs). Photographs are representative of data from (Objectives X10) 3 donors. B) Photographs of hMSCs after methylene blue and alizarin red S staining, 21 days following osteogenic differentiation after irradiation (0-6-10 Gy). Methylene blue staining was used to assess hMSCs morphology in undifferentiated (ND-hMSCs) and osteogenically-differentiated cells (OS-hMSCs) after irradiation. Alizarin red S staining was used to detect the presence of calcium deposition in undifferentiated (ND-hMSCs) and osteogenically- differentiated cells (OS-hMSCs) after X-rays irradiation. Representative photographs (Objectives X10) of 3 donors. C) Real-time RT-PCR analysis of relative mRNA expression of the indicated genes (ALP-alkaline phosphatase, OPN-osteopontin, BSP-Bone sialoprotein, SOX9-Sox9 transcription factor, OCN-Osteocalcin, RUNX2-runt-related transcription factor 2, COL1A1-type I collagen, COL10A1-type X collagen, COL2A1-type II collagen) in undifferentiated hMSCs (ND-hMSCs) and osteogenically differentiated hMSCs (OS-hMSCs) after irradiation (0-6-10 Gy). Gene expression is normalized against the endogenous reference gene, RPL13A. Results are expressed as relative mRNA expression as compared to the control condition (undifferentiated hMSCs, 0 Gy condition) set at the arbitrary unit of 1 Box and whiskers plots (Min to Max. Bar at median and + at mean) of data derived from 5 donors. Statistical analysis (One-Way ANOVA, with Tukey post-test) was performed and statistical significance is shown with, * for p<0.05, and ** for p< 0.01. D) Western blot analysis of type II collagen, type I collagen, and GAPDH in undifferentiated (ND-hMSCs) and osteogenically differentiated cells (OS-hMSCs) after irradiation. E) Real-time RT-PCR analysis of expression levels of the indicated genes (MMP-1, MMP-3, and MMP-13) in undifferentiated hMSCs (ND-hMSCs) and osteogenically differentiated hMSCs (CH 3D-hMSCs) after irradiation (0-6-10 Gy). Gene expression is normalized against the endogenous reference gene, RPL13A. Results are expressed as relative mRNA expression as compared to the control condition (undifferentiated hMSCs, 0 Gy condition) set at the arbitrary unit of 1. Box and whiskers (Min to Max. Bar at median and + at mean) of 4 independent experiments. Statistical analysis (One-Way ANOVA, with Tukey post-test) were performed and statistical significance is shown with, * for p<0.05, and ** for p< 0.01.
Mentions: Osteogenic differentiation was induced after X-irradiation and assessed 21 days later. As seen in light microscopy photographs (Fig 3A), dense, refractile deposits, indicative of mineralization were seen in osteogenically differentiated hMSCs but not in the undifferentiated hMSCs (compare ND-hMSCs and OS-hMSCs at 0 Gy). In addition, a dose-dependent increase in the density of these deposits was observed in the irradiated and osteogenically differentiated hMSCs (OS-hMSCs), with denser deposits being observed after 10 Gy compared to 6 Gy. In order to specifically assess that these deposits were genuine calcium deposits, we performed an Alizarin red solution staining. Methylene blue staining was also performed in order to show the morphological changes associated with osteoblastic differentiation. As seen in Fig 3B (methylene blue), osteogenically differentiated hMSCs (OS-hMSCs) presented a more cuboidal morphology, characteristic of osteoblasts than the undifferentiated hMSCs, indicating proper differentiation induction. Moreover, no alizarin red staining was observed in the undifferentiated hMSCs, while osteogenically differentiated hMSCs exhibited a strong red staining indicative of calcium deposition and osteogenic differentiation. hMSCs, that had been osteogenically differentiated after 6 Gy and 10 Gy, despite showing a lower density than the undifferentiated hMSCS, presented a more pronounced alizarin red staining compared to the mock-irradiated OS-hMSCs, and displayed mineralized nodules, indicative of increased differentiation after 6 and 10 Gy.

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