Radiation rescue: mesenchymal stromal cells protect from lethal irradiation

Abstract

Background: Successful treatment of acute radiation syndromes relies on immediate supportive care. In patients with limited hematopoietic recovery potential, hematopoietic stem cell (HSC) transplantation is the only curative treatment option. Because of time consuming donor search and uncertain outcome we propose MSC treatment as an alternative treatment for severely radiation-affected individuals.

Methods and findings: Mouse mesenchymal stromal cells (mMSCs) were expanded from bone marrow, retrovirally labeled with eGFP (bulk cultures) and cloned. Bulk and five selected clonal mMSCs populations were characterized in vitro for their multilineage differentiation potential and phenotype showing no contamination with hematopoietic cells. Lethally irradiated recipients were i.v. transplanted with bulk or clonal mMSCs. We found a long-term survival of recipients with fast hematopoietic recovery after the transplantation of MSCs exclusively without support by HSCs. Quantitative PCR based chimerism analysis detected eGFP-positive donor cells in peripheral blood immediately after injection and in lungs within 24 hours. However, no donor cells in any investigated tissue remained long-term. Despite the rapidly disappearing donor cells, microarray and quantitative RT-PCR gene expression analysis in the bone marrow of MSC-transplanted animals displayed enhanced regenerative features characterized by (i) decreased proinflammatory, ECM formation and adhesion properties and (ii) boosted anti-inflammation, detoxification, cell cycle and anti-oxidative stress control as compared to HSC-transplanted animals.

Conclusions: Our data revealed that systemically administered MSCs provoke a protective mechanism counteracting the inflammatory events and also supporting detoxification and stress management after radiation exposure. Further our results suggest that MSCs, their release of trophic factors and their HSC-niche modulating activity rescue endogenous hematopoiesis thereby serving as fast and effective first-line treatment to combat radiation-induced hematopoietic failure.

Figure 1. Mouse MSCs rescue mice after total body irradiation.

Transplantation of bulk mMSCs led to a normalization of the peripheral white blood cell count within 4 weeks. Thrombocyte recovery needed approx. 8 weeks for normalization. Thus, results are comparable to blood recovery after HSC transplantation.

Figure 2. Integration site pattern of clonal mMSCs.

The clonal mMSCs were investigated using LM-PCR. Each of the clones represents a specific pattern of integration sites. Bands marked with red asterix were subjected to sequencing for further characterization (see Table S1). IC, internal control.

Figure 3. Differentiation potential of mMSCs.

Mouse MSCs were generated from male C57BL/6J bone marrow and expanded for 9 passages. Expanded mMSC were retrovirally transduced with eGFP (bulk) and cloned. Five clones with sufficient growth were selected and further expanded. They differed regarding their morphology and growth pace. Cells from passages 14–16 were induced to differentiate into adipogenic, osteogenic and chondrogenic cells. All clones and the parental bulk cells demonstrated three-lineage differentiation capability. Noninduced controls were negative for the respective stainings (not shown).

Figure 4. Donor mMSCs are not detectable long-term.

(a) Tracking of eGFP-labeled clonal IXH8 donor mMSCs after transplantation revealed a fast decrease in PB. Within 8 hours, approx. 2% were quantified in PB and none after 10 days (n = 8 for each time point). Insert: mMSCs accumulated in lungs (Lu) within 24 h and disappeared within 10 days (not shown). Spleen (Sp), liver (Li) and BM were negative at d1. (b) Based on standard dilutions (filled symbols), no eGFP signals (open symbols) above the assay's detection limit of approx. 0.5% were detected in the BM of long-term survivors reconstituted with mMSCs of clone IXH8 (dashed line, detection limit of qPCR). nd, not detected.

Figure 5. Spectral karyotyping of mMSCs.

(a) Shown is the SKY analysis of clone IXH8. SKY analysis revealed clonal structural and numerical chromosomal alterations as demonstrated in the spectral image of a representative diploid metaphase. (b) In most metaphases, we observed a hypertriploid (representative metaphase shown here) to an almost hypotetraploid chromosome complement with loss of the Y-chromosome in all metaphases analyzed.

Figure 6. Transplantation of mMSCs leads to gene expression changes in BM supporting rescue of endogenous hematopoiesis.

(a) Bootstrap hierarchical clustering of all 20K genes depicted highly stable clusters for HSC/BM vs. MSC. (b) Heat map clustering using average distance and Manhattan metric for 2 microarrays per group hybridized with RNA from pooled BM (HSC: 2 animals/array, MSC: 3 animals/array) is shown for genes summarized in Table 3. (c) Gene expression ratios of selected genes using BM of mice 21 days after transplantation with MSCs (n = 9) or HSCs (n = 10) were investigated for independent cohorts (grey columns: microarray data; white columns: quantitative PCR). Shown are mean ratios ± SEM. P<0.005. Suggested functions of validated genes are shown in italics.

Figure 7. Ectopic ossicles in mice lungs after i.v. transplantation of bulk mMSCs.

Mice subjected to total body irradiation and transplanted i.v. with syngeneic MSCs were analyzed after 7 months. In lungs, fibrotic lesions were detected with HE staining (a) which showed the typical dark precipitates in von-Kossa stainings (b) admixed with large Collagen I-positive areas (c) suggesting bone and cartilage containing ossicles.