Adipose-Derived Mesenchymal Stem Cells with Potential to Ameliorate Platelet Recovery and Enhance Megakaryopoiesis in a Mouse Model of Radiation-Induced Thrombocytopenia Via the PI3k/Akt Pathway

*XHZ and JMZ contribute equally to this work

# Co-correspondence to: Xiao-hui Zhang and Xiao-Jun Huang

E-mail: zhangxh100@sina.com

Introduction: Substantial damage to the bone marrow can be caused by exposure to radiation, which can then develop into pancytopenia, especially severe thrombocytopenia. Severe thrombocytopenia can be a major, life-threatening event in untreated individuals. Recent studies have highlighted the role that radiation-induced thrombocytopenia plays in radiation mortality. However, studies focused on mitigating radiation-induced thrombocytopenia have rarely been reported. Among all alternative therapies available to patients experiencing radiation-induced thrombocytopenia, platelet transfusion is most effective. As a supportive therapy, platelet transfusion cannot substantially reconstitute the damaged hematopoietic properties of the bone marrow. Adipose-derived mesenchymal stem cells (ADSCs) are capable of migrating to injured tissue sites for damage repair. Moreover, it was demonstrated that ADSCs could support hematopoiesis both in vitro and in vivo. However, the therapeutic effects of ADSCs in radiation-induced thrombocytopenia as well as the underlying mechanism remain unknown. In this study, we hypothesized that administration of ADSCs may mitigate thrombocytopenia after radiation exposure. We investigated the in vivo impact of ADSCs on megakaryopoiesis and platelet recovery, and whether this amelioration effect was mediated through the activation of PI3K/Akt pathway in irradiated mice.

Methods: The mouse model of radiation-induced thrombocytopenia was established by first irradiating mice with a 4Gy dose. Then, 15 mice were immediately injected with suspended ADSCs (1×10⁶cells in 0.3 ml) and another 15 mice with equivalent saline. Ten unirradiated mice served as a control group. Platelet counts and white blood cell (WBC) counts in the peripheral blood were detected every week. Total colony formation units (CFU), megakaryocyte colony formation units (MK-CFU) and CD41+ cells in the bone marrow were assessed 21 days after irradiation. Bone marrow cellularity was determined by hematoxylin and eosin (HE) staining, and apoptosis was detected by terminal-deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay. Western blots were performed with anti-phosphorylated Akt, anti-Bcl-2 and anti-bax antibodies.

Results: ADSCs obtained from the inguinal adipose exhibited a fibroblast-like morphology. Cultured cells were positive for CD29 (99.79%) and CD90 (97.82%), but not CD34 (3.08%), CD45 (1.04%) and CD31 (3.08%).

Radiation markedly reduced peripheral blood counts, with a nadir on day 7. Recovery of both platelets (546.33±62.99 vs 375.48±50.33×10⁹/L, P<0.05) and WBCs (4.23±0.51 vs 2.46±3.10×10⁹/L, P<0.05) were better in the ADSCs-treated group compared with the saline group, respectively, 21 days after irradiation. A significant increase in total CFU (34.55±4.21 vs 12.86±2.15, P<0.05) and MK-CFU (6.28±0.74 vs 2.54±0.39, P<0.05) after irradiation were observed in the ADSCs group compared with the saline group, respectively. Further, the proportion of CD41+ cells in the ADSCs group was significantly higher than that in the saline group (4.2%±0.54% vs 1.21%±0.11%, P<0.05).

A radioprotective effect was shown in the ADSCs-treated group, especially in the megakaryocytic lineage, by HE staining. In the ADSCs group, the number of apoptotic cells was significantly lower than that in the saline group (3.52±0.42 vs 13.48±2.15 per field, P<0.05). Administration of ADSCs up-regulated protein expression of phosphorylated Akt and Bcl-2, whereas the expression of Bax, a protein related to apoptosis, was significantly lower in the ADSCs group.

Conclusion:For the first time, we demonstrated that ADSCs were capable of promoting platelet recovery and improving megakaryopoiesis in irradiated mice. This protective function of ADSCs is likely to be mediated via the PI3K/Akt pathway, which would thus provide a new therapeutic alternative for mitigating radiation-induced thrombocytopenia.