Mesenchymal stem cells show radioresistance in vivo

Abstract

Irradiation impacts on the viability and differentiation capacity of tissue-borne mesenchymal stem cells (MSC), which play a pivotal role in bone regeneration. As a consequence of radiotherapy, bones may develop osteoradionecrosis. When irradiating human bone-derived MSC in vitro with increasing doses, the cells' self-renewal capabilities were greatly reduced. Mitotically stalled cells were still capable of differentiating into osteoblasts and pre-adipocytes. As a large animal model comparable to the clinical situation, pig mandibles were subjected to fractionized radiation of 2 χ 9 Gy within 1 week. This treatment mimics that of a standardized clinical treatment regimen of head and neck cancer patients irradiated 30 χ 2 Gy. In the pig model, fractures which had been irradiated, showed delayed osseous healing. When isolating MSC at different time points post-irradiation, no significant changes regarding proliferation capacity and osteogenic differentiation potential became apparent. Therefore, pig mandibles were irradiated with a single dose of either 9 or 18 Gy in vivo, and MSC were isolated immediately afterwards. No significant differences between the untreated and 9 Gy irradiated bone with respect to proliferation and osteogenic differentiation were unveiled. Yet, cells isolated from 18 Gy irradiated specimens exhibited a reduced osteogenic differentiation capacity, and during the first 2 weeks proliferation rates were greatly diminished. Thereafter, cells recovered and showed normal proliferation behaviour. These findings imply that MSC can effectively cope with irradiation up to high doses in vivo. This finding should thus be implemented in future therapeutic concepts to protect regenerating tissue from radiation consequences.

Fig 1

Irradiation of in vitro cultivated human mesenchymal stromal cells derived from cancellous bone of the iliac crest. (A) Irradiation of proliferating cells with the indicated dosage showed an impact on cell cycle progression of the surviving cell fraction and cell survival (B). (C) Colony formation was decreased after irradiation treatment (assessed in duplicates; a representative example displayed in the lower part of panel C). (D) High-power photomicrographs depicting cells after irradiation (doses in Gy) as indicated. (E) Adipogenic differentiation potential decreased after irradiation in vitro at indicated dosages (measured in triplicates; representative examples of Oil Red stained photomicrographs in the lower part of E); for specification of arbitrary units, see Materials and methods. (F) Osteogenic differentiation capacity was enhanced after irradiation up to doses as high as 12 Gy, shown as a set of representative examples of Alizarin Red stained photomicrographs. Experimental results in bar graphs are shown as mean ± standard deviation.

Fig 2

Properties of in vitro cultured porcine MSC. Fibroblastoid cells, which exhibited firm plastic adherence (A), clonogenic growth (B), a respective immune phenotype (C) and tri-lineage differentiation capacity (D–F), were isolated from mandibular bone, bone marrow and periosteum as well as from cancellous bone of the iliac crest. (C) Immune phenotyping of porcine MSC was performed taking only 7-aminoactinomycin D-negative cells into account; negative controls comprise unstained cells. Fractions highlighted in grey comprise cells stained solely with 7-AAD; fraction shown in red are cells stained with the indicated monoclonal antibody. (D) Differentiation capacity was shown for adipogenic potential (adipo), both applying histological staining with oil red, and quantitative RT-PCR analysis for the molecular marker aP2 and un-induced control cells (con). (E) Osteogenic potential (osteo) was proven by staining cultures with alizarin red S and the expression of the osteogenic marker osteocalcin (oc). (F) Chondrogenic differentiation (chond) could be demonstrated in pellet culture: in case of induced MSC the aggregates expanded in size (upper left panel), and stimulated MSC activated chondrogenic markers such as sox 9 and aggrecan (agg) (upper row, centre and right side). Paraffin sections of the aggregate culture efficiently bound the histological dye alcian blue (lower row, left and centre panels) and showed collagen II expression after immunohistochemical evaluation with a specific antibody. Experimental results in bar graphs are shown as mean ± standard deviation.

Fig 3

Culture and in vitro osteogenic differentiation of primary porcine mesenchymal stromal cells. (A, B) Osteogenic differentiation potential decreased after irradiation in vitro at the indicated dosage (arbitrary units were distinguished as depicted left side to the graph depicted in (B) grade 3–more than 60% of cells engulfed by mineralized matrix; grade 2—40–60%; grade 1—less than 40%; grade 0—no differentiation in reference to negative control scale bar equals 1 cm. Experimental results in bar graphs are shown as mean ± standard deviation.

Fig 4

Fracture healing in irradiated mandible of Sus scrofa domestica. Bone healing was investigated 8 weeks after a fracture gap was set. Samples (A) and (C) were irradiated, (B) and (D) are untreated controls. Representative examples of slow or poor healing (A, B) juxtaposed to a more rapid course (C, D) in both irradiated (A, C) and control mandibular bone; dashed line marks the former edge of the fracture gap (FG); connective tissue (CT) stains blue, local bone is labelled LB. Experimental results in bar graphs are shown as mean ± standard deviation.

Fig 5

Properties of primary porcine mesenchymal stromal cells isolated from the mandible directly after irradiation with the indicated effective biological dosage. (A) The proliferation potential was monitored in long-term culture. (B) Colony formation was accounted in low-density secondary culture. (C) After irradiation and subsequent cultivation in the presence of osteogenic induction medium, the differentiation potential was assessed in triplicates (for grading, see left panel). For specification of arbitrary units, see Materials and methods. Experimental results in bar graphs are shown as mean ± standard deviation.

Fig 6

Clonogenic growth of porcine mesenchymal stromal cells isolated from the mandible after fractionated irradiation with 2 χ 9 Gy. (A) Colony formation of primary cultivated cells isolated 4–6 weeks post-irradiation and (B) integration of data accounted from all primary cultures isolated at the latter time points. (C) Integration of data accounted from all assessments of osteogenic differentiation with MSC isolated at time points indicated in (A). Specification of arbitrary units, see Materials and methods. Experimental results in bar graphs are shown as mean ± standard deviation.

Fig 7

Proliferation potential of porcine mesenchymal stromal cells. (A) Growth kinetics of cells were isolated from trabecular bone of the mandible 4 weeks after fractionated irradiation with 2 χ 9 Gy, and grown in long-term culture (representative examples). As controls, MSC were also harvested from trabecular bone as well as bone marrow of unirradiated animals. (B) Proliferation index of cells isolated 4–6 weeks post-irradiation during their early stages of long-term cultures and integration of data accounted from all long-term cultivations; white bars show unirradiated controls, black bars are MSC isolated after radiation treatment with 18 Gy. (C) Integration of data accounted from all long-term cultures cultures isolated at the latter time points as indicated. Experimental results in bar graphs are shown as mean ± standard deviation.