Marrow Stromal Cells (MSC) Rescue Hematopoiesis in Lethally Irradiated Mice Despite Rapid Clearance After Infusion,

Abstract 3406

Ionizing radiation causes dose dependent damages in many organs with most pronounced effects on those with high proliferative potential such as the hematopoietic system and gastrointestinal tract. Lympho-hematopoietic failure is often the cause of death following moderate to severe exposures to radiation. Effects of low to moderate (sub-lethal) doses of radiation can be mitigated by cytokines such as granuclocyte colony stimulating factor (GCSF) or Flt-3 ligand; higher doses of radiation require a new hematopoietic system by stem cell transplantation (SCT). As SCT for large numbers of radiation victims is not only costly but impractical following mass casualties (given the need for tissue matching prior to transplantation), there is considerable interest in cellular therapies that can be rapidly expanded, have a long shelf life and do not require tissue matching. Mesenchymal stromal cells (MSC, also referred to as mesenchymal stem cells) have been proposed as one such cellular therapy that could improve survival after moderate to high doses of radiation, but direct evidence for such a clinical benefit is scant in pre-clinical models. To determine if infusion of MSCs following ionizing radiation may rescue hematopoiesis after lethal irradiation in the murine model, we first determined the LD50 (lethal dose 50 or the dose at which 50% of animals survive without specific intervention) in C57/BL6 mice to be between 600 and 700 cGy when a X-Ray irradiator (RS2000) was used as the source of ionizing radiation. We then radiated adult female C57/Bl6 mice (age 6 to 8 weeks) with 700 cGy followed by infusion of either pooled immortalized MSC clones (henceforth referred to as cMSC, 1×10⁶ cells each, n=19) or primary MSC (referred to as pMSC, 1×10⁶ cells each, n=20) by tail vein injection 2 hours later. MSC were syngeneic to the recipients. Control mice received equal volume of PBS (n=21). Survival at the end of 7 weeks after radiation was determined using log-rank test which showed that animals that received either cMSC or pMSCs had significantly improved survival rates (p values of 0.017 and 0.041 respectively) when compared to the control animals that received only PBS (Figure 1 ).

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The precise mechanism of action of MSCs after systemic infusion in various tissue injury models is currently undefined. Although trans-differentiation to host tissues has been proposed as one potential mechanism, recent reports have suggested that there is little evidence of persistence of infused MSC after the initial few days in the target tissues making trans-differentiation unlikely. Hence we determined the in vivo distribution kinetics of infused MSC by three techniques: whole-body bioluminescent imaging. (BLI), immune histochemistry (IHC) and quantitative real time polymerase chain reaction (q RT PCR). MSC were labeled with firefly luciferase (ffLuc) using a lentiviral vector and infused to adult female recipients after they were administered 700 cGy radiation (1×10⁶ cells each, n=7). In all mice, strong bioluminescent signals were detected from the chest region at 4 hours after infusion, suggesting an accumulation of infused MSCs in the lungs. The signals rapidly decreased during the first 24 hours, and no bioluminescent signal was detected at 72 hours after infusion. No signals were detectable from other organs (liver, spleen or long bones) at any time point. Ffluc could not be reliably detected in tissues by IHC. Detection of luciferase transcripts in different tissues (lungs, hearts, spleens, livers, guts, muscles, and bone marrow) was performed by quantitative RT PCR at days 1, 4 and 7 (n = 6 each) following infusion. Ffluc transcripts were reliably detected in all animals only in the lungs at 24 hours after MSC infusion, confirming the BLI results. At subsequent time points, ffluc transcripts were detectable at very low levels in a variable proportion of animals from various tissues. In the absence of reliable BLI signals from these extra-pulmonary tissues, we interpret the presence of low levels of ffluc transcripts in these tissues as to have arisen from unviable cells or circulating RNA. Together, these results show that both immortalized MSCs and primary MSCs improve hematopoietic recovery after lethal ionizing radiation, but the infused cells are mostly filtered out by the lungs and their beneficial effect is likely mediated by indirect mechanisms (secondary effector cells in the lungs or secreted cytokines).