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Human bone marrow stromal cells display variable anatomic site-dependent response and recovery from irradiation.

ABSTRACT: Orofacial bone is commonly affected by osteoradionecrosis (ORN) during head and neck cancer radiotherapy possibly due to interactions of several factors including radiation damage to resident bone marrow stromal cells (BMSCs). Irradiation causes DNA damage, triggers p53-dependent signalling resulting in either cell-cycle arrest or apoptosis. In same individuals, disproportionately higher rapid growth of orofacial BMSCs relative to those of axial/appendicular bones suggests their response to radiation is skeletally site-specific. We hypothesised that survival and osteogenic recovery capacity of irradiated human BMSCs is site-dependent based on anatomic skeletal site of origin.Early passage BMSCs from maxilla, mandible and iliac crest of four normal volunteers were exposed to 2.5 to 10 Gy gamma radiation to evaluate clonogenic survival, effects on cell cycle, DNA damage, p53-related response and in vivo osteogenic regenerative capacity.Orofacial bone marrow stromal cells (OF-MSCs) survived higher radiation doses and recovered quicker than iliac crest (IC-MSCs) based on clonogenic survival, proliferation and accumulation in G0G1 phase. Post-irradiation p53 level was relatively unchanged but expression of p21, a downstream effector was moderately increased in OF-MSCs. Re-establishment of in vivo bone regeneration was delayed more in irradiated IC-MSCs relative to OF-MSCs.Effect of irradiation on human BMSCs was skeletal site-specific with OF-MSCs displaying higher radio-resistance and quicker recovery than IC-MSCs.

SUBMITTER: Damek-Poprawa M

PROVIDER: S-EPMC2886662 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Human bone marrow stromal cells display variable anatomic site-dependent response and recovery from irradiation.

Damek-Poprawa Monika M Stefanik Derek D Levin Lawrence M LM Akintoye Sunday O SO

Archives of oral biology 20100407 5

<h4>Objectives</h4>Orofacial bone is commonly affected by osteoradionecrosis (ORN) during head and neck cancer radiotherapy possibly due to interactions of several factors including radiation damage to resident bone marrow stromal cells (BMSCs). Irradiation causes DNA damage, triggers p53-dependent signalling resulting in either cell-cycle arrest or apoptosis. In same individuals, disproportionately higher rapid growth of orofacial BMSCs relative to those of axial/appendicular bones suggests the ...[more]

PMID: 20378097

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Project description:Cell therapy using multipotent mesenchymal stromal cells (MSCs) is of high interest in various indications. As the pleiotropic effects mediated by MSCs rely mostly on their unique secretory profile, long-term persistence of ex-vivo-expanded cells in the recipient may not always be desirable. Irradiation is a routine procedure in transfusion medicine to prevent long-term persistence of nucleated cells and could therefore also be applied to MSCs. We have exposed human bone-marrow-derived MSCs to 30 or 60 Gy of ?-irradiation and assessed cell proliferation, clonogenicity, differentiation, cytokine levels in media supernatants, surface receptor profile, as well as expression of proto-oncogenes/cell cycle markers, self-renewal/stemness markers, and DNA damage/irradiation markers. Irradiated MSCs show a significant decrease in proliferation and colony-forming unit-fibroblasts. However, a subpopulation of surviving cells is able to differentiate, but is unable to form colonies after irradiation. Irradiated MSCs showed stable expression of CD73 and CD90 and absence of CD3, CD34, and CD45 during a 16-week follow-up period. We found increased vascular endothelial growth factor (VEGF) levels and a decrease of platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB in culture media of nonirradiated cells. Irradiated MSCs showed an inverse pattern, that is, no increase of VEGF, and less consumption of PDGF-AA and PDGF-AB/BB. Interestingly, interleukin-6 (IL-6) levels increased during culture regardless of irradiation. Cells with lower sensitivity toward ?-irradiation showed positive ?-galactosidase activity 10 days after irradiation. Gene expression of both irradiated and nonirradiated MSCs 13-16 weeks after irradiation with 60 Gy predominantly followed the same pattern; cell cycle regulators CDKN1A (p21) and CDKN2A (p16) were upregulated, indicating cell cycle arrest, whereas classical proto-oncogenes, respectively, and self-renewal/stemness markers MYC, TP53 (p53), and KLF4 were downregulated. In addition, DNA damage/irradiation markers ATM, ATR, BRCA1, CHEK1, CHEK2, MDC1, and TP53BP1 also mostly showed the same pattern of gene expression as high-dose ?-irradiation. In conclusion, we demonstrated the existence of an MSC subpopulation with remarkable resistance to high-dose ?-irradiation. Cells surviving irradiation retained their trilineage differentiation capacity and surface marker profile but changed their cytokine secretion profile and became prematurely senescent.

Project description:BackgroundBone marrow stromal cells (BMSCs) are extensively used in regeneration therapy and cytology experiments simulate how BMSCs respond to radiation. Due to the small number and the heterogeneity of primary isolated BMSCs, extensive in vitro expansion is usually required before application, which affects the cellular characteristics and gene expression of BMSCs. However, whether the radiation response of BMSCs changes during in vitro expansion is unclear.MethodsIn this study, BMSCs were passaged in vitro and irradiated at passage 6 (P6) and passage 10 (P10). Then, apoptosis, the cell cycle, senescence, the cytokine secretion and the gene expression profile were analysed for the P6, P10, and non-irradiated (control) BMSCs at different post-irradiation time points.ResultsThe P6 BMSCs had a lower percentage of apoptotic cells than the P10 BMSCs at 24 and 48 h post-irradiation but not compared to that of the controls at 2 and 8 h post-irradiation. The P6 BMSCs had a lower percentage of cells in S phase and a higher percentage in G1 phase than the P10 BMSCs at 2 and 8 h post-irradiation. The radiation had similar effects on the senescent cell level and impaired immunomodulation capacity of the P6 and P10 BMSCs. Regardless of whether they were irradiated, the P6 and P10 BMSCs always expressed a distinctive set of genes. The upregulated genes were enriched in pathways including the cell cycle, DNA replication and oocyte meiosis. Then, a subset of conserved irradiation response genes across the BMSCs was identified, comprising 12 differentially upregulated genes and 5 differentially downregulated genes. These genes were especially associated with the p53 signaling pathway, DNA damage and DNA repair. Furthermore, validation experiments revealed that the mRNA and protein levels of these conserved genes were different between the P6 and P10 BMSCs after irradiation. Weighted gene co-expression network analysis supported these findings and further revealed the effects of cell passage on the irradiation response in BMSCs.ConclusionThe results indicated that cell passage in vitro affected the irradiation response of BMSCs via molecular mechanisms that mediated differences in apoptosis, the cell cycle, senescence and the cytokine secretion. Thus, accurate cell passage information is not only important for transplantation therapy but also for future studies on the radiation response in BMSCs.

Project description:BACKGROUND:Radiotherapy remains one of the cornerstones to improve the outcome of colorectal cancer (CRC) patients. Radiotherapy of the CRC not only help to destroy cancer cells but also remodel the tumour microenvironment by enhancing tumour-specific tropism of bone marrow-derived mesenchymal stromal cell (BM-MSC) from the peripheral circulation. However, the role of local MSCs and recruited BM-MSC under radiation were not well defined. Indeed, the functions of BM-MSC without irradiation intervention remained controversial in tumour progression: BM-MSC was previously shown to modulate the immune function of major immune cells, resulting in an impaired immunological sensitivity and to induce an increased risk of tumour recurrence. In contrast, it could also secrete various cytokines and possess anticancer effect. METHODS:Three co-cultivation modules, 3D culture modules, and cancer organoids were established. The induction of cytokines secretion in hBM-MSCs after irradiation was analysed by ELISA array and flow cytometry. AutoMac separator was used to separate hBM-MSC and CRC automatically. Cells from the co-cultured group and the control group were then irradiated by UV-C lamp and X-ray. Proliferation assay and viability assay were performed. RESULTS:In this study, we show that BM-MSCs can induce the EMT progression of CRC cells in vitro. When irradiated with low doses of ultraviolet radiation and X-rays, BM-MSCs show an anti-tumour effect by secreting certain cytokine (TNF-?, IFN-?) that lead to the inhibition of proliferation and induction of apoptosis of CRC cells. This was further verified in a 3D culture model of a CRC cell in vitro. Furthermore, irradiation on the co-culture system induced the cleavage of caspase3, and attenuated the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase in cancer cells. The signal pathways above might contribute to the cancer cell death. CONCLUSIONS:Taken together, we show that BM-MSC can potentially promote the effect of radiotherapy in CRC.

Project description:BACKGROUND:Bone marrow aspirate is a primary source for cell-based therapies with increasing value in the world of orthopaedic surgery, especially in revision cases of tendon and ligament repairs. However, cells within peritendinous structures, such as the paratenon and surrounding bursa, contribute to the native tendon-healing response and offer promising cell populations for cell-based repair strategies. Therefore, the purpose of this study is to investigate the efficacy of cells derived from human subacromial bursa as compared with the current gold standard, bone marrow stromal cells (BMSCs), for tendon repairs in an established in vivo immunodeficient murine patellar tendon defect model. HYPOTHESIS:Subacromial bursal cells will show superior survival and engraftment into the host tissue as compared with BMSCs. STUDY DESIGN:Controlled laboratory study. METHODS:Human subacromial bursal and bone marrow aspirate were harvested from the same donor undergoing rotator cuff repair. Cells were transfected with a fluorescent lentiviral vector to permanently label the cells, encapsulated into fibrin gel, and implanted into bilateral full-length central-width patellar tendon defects of immunodeficient mice. Additional surgery was performed on control mice comparing fibrin without cells and natural healing. At the time of sacrifice, all limbs were scanned on a multiphoton microscope to monitor the engraftment of the human donor cells. Afterward, limbs were assigned to either immunohistochemical or biomechanical analysis. RESULTS:As compared with BMSCs, implanted subacromial bursal cells displayed superior tissue engraftment and survival. The main healing response in this defect model was the creation of new healing tissue over the anterior surface of the defect space. The implantation of cells significantly increased the thickness of the anterior healing tissue as compared with control limbs that did not receive cells. Cell proliferation was also increased in limbs that received implanted cells, suggesting that the donor cells stimulated a more robust healing response. Finally, these changes in the healing response did not lead to significant changes in mechanical properties. CONCLUSION:The subacromial bursa, while often removed during rotator cuff repair, may harbor a more suitable cell source for tendon repair than BMSCs, as bursal cells display superior engraftment and survival in tendon tissue. In addition, the subacromial bursa may be a more accessible cell source than bone marrow aspirate. CLINICAL RELEVANCE:The subacromial bursa contains a cell population that responds to tendon injury and may provide a more optimal cell source for tendon repair and regeneration strategies. Therefore, cells could be harvested from this tissue in the future, as opposed to the current practice of bursectomy and debridement.

Project description:BackgroundDirect bone marrow injection of cells into murine marrow cavities is used in a range of cell characterization assays and to develop disease models. While human bone marrow-derived stromal cells (hBMSC, also known as mesenchymal stem cells (MSC)) are frequently described in therapeutic applications, or disease modeling, their behavior following direct injection into murine bone marrow is poorly characterized. Herein, we characterized hBMSC engraftment and persistence within the bone marrow of NOD-scid interleukin (IL)-2γ-/- (NSG) mice with or without prior 2 Gy total-body γ-irradiation of recipient mice.MethodsOne day after conditioning NSG mice with sublethal irradiation, 5 × 105 luciferase (Luc) and green fluorescent protein (GFP)-expressing hBMSC (hBMSC-Luc/GFP) were injected into the right femurs of animals. hBMSC-Luc/GFP were tracked in live animals using IVIS imaging, and histology was used to further characterize hBMSC location and behavior in tissues.ResultshBMSC-Luc/GFP number within injected marrow cavities declined rapidly over 4 weeks, but prior irradiation of animals delayed this decline. At 4 weeks, hBMSC-Luc/GFP colonized injected marrow cavities and distal marrow cavities at rates of 2.5 ± 2.2% and 1.7 ± 1.9% of total marrow nucleated cells, respectively in both irradiated and non-irradiated mice. In distal marrow cavities, hBMSC were not uniformly distributed and appeared to be co-localized in clusters, with the majority found in the endosteal region.ConclusionsWhile significant numbers of hBMSC-Luc/GFP could be deposited into the mouse bone marrow via direct bone marrow injection, IVIS imaging indicated that the number of hBMSC-Luc/GFP in that bone marrow cavity declined with time. Irradiation of mice prior to transplant only delayed the rate of hBMSC-Luc/GFP population decline in injected femurs. Clusters of hBMSC-Luc/GFP were observed in the histology of distal marrow cavities, suggesting that some transplanted cells actively homed to distal marrow cavities. Individual cell clusters may have arisen from discrete clones that homed to the marrow, and then underwent modest proliferation. The transient high-density population of hBMSC within the injected femur, or the longer-term low-density population of hBMSC in distal marrow cavities, offers useful models for studying disease or regenerative processes. Experimental designs should consider how relative hBMSC distribution and local hBMSC densities evolve over time.

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Human bone marrow stromal cells display variable anatomic site-dependent response and recovery from irradiation.

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Human bone marrow stromal cells display variable anatomic site-dependent response and recovery from irradiation.

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