3.1 STS stimulated the generation of BMSCs-derived ABs
We used STS (500 nM), a widely used apoptosis inducer, to stimulate BMSCs apoptosis. TEM showed shrinkage of the cell, breakage of the nucleus and cytoplasmic organelles, and cell membrane detachment and blebbing (Fig. 1A), which are typical features of apoptotic cells. Light microscopic analyses showed obvious morphological changes with the prolong of STS treatment (Fig. 1B). FCM analyses (Fig. 1C-D) of Annexin V/ PI - stained BMSCs showed the percentage of apoptotic cells increased gradually with the prolongation of STS treatment from 49.8% at 2 h to 75.4% at 12 h, suggesting STS induced BMSCs apoptosis in a time-dependent manner. Besides, the most cells after STS treated for 4 h were undergoing early apoptosis compared with other groups. According to the percentage of positive cells, we defined four stages of apoptosis: the early (STS treatment for 2 h), the middle (STS treatment for 4 h), the middle-late (STS treatment for 6 h), and late (STS treatment for 12 h) stage.
Then we used ultracentrifugation to extracted ABs from apoptotic BMSCs for identification as previously described (Fig. 2A). TEM analysis confirmed the presence of round-shaped vesicles surrounded by a bilayer membrane (Fig. 2B). Ultrathin sections of ABs imaged by TEM showed nucleus fragments, cytoplasmic organelles, and autophagosomes, which were also presented in apoptotic cells. Western blot analysis showed that ABs contained specific markers including histone 3 (H3), and Complement C3b, and lack the exosome markers (CD63) (Fig. 2C). Then, we detect the expression of Annexin V in ABs. The ratio of Annexin V+ in ABs was up to 81.3% (Fig. 2D). The protein content showed that the longer the STS treatment time was, the higher ABs protein was (Fig. 2I). The results showed the ABs we used in this study were vesicles as approximately 1–4 µm in size, with Annexin V positive and H3, C3b expression.
3.2 ABs from the middle stage of apoptosis are the most potent to induce proliferation of BMSCs
Next, we determined the effect of ABs on the recipient BMSCs. Firstly, we examined whether recipient BMSCs could engulf ABs in the micro-environment. After BMSCs co-cultured with ABs for 24 h, immunofluorescent staining showed that BMSCs could successfully uptake exogenous PKH67-labeled ABs (Fig. 3).
To compare the proliferation potency of ABs from different stages of apoptosis on BMSCs, the growth curves determined by the CCK-8 assay (Fig. 4A) showed that ABs from the middle stage of apoptosis promoted mouse BMSCs (m-BMSCs) proliferation, while ABs from other stages of apoptosis has no obvious effect. To confirm that the effect of ABs is not species-specific, we also repeated the above experiment using BMSCs and ABs derived from rats. Although slightly different compared with m-BMSCs, the CCK-8 assay on rat-BMSCs (r-BMSCs) (Fig. 4B) showed that ABs from all stages of apoptosis promoted the growth and proliferation of r-BMSCs. But it should be noted that ABs from the middle stage of apoptosis had the strongest ability to promote the proliferation of r-BMSCs. In brief, ABs from the middle stage of apoptosis had the highest ability to promote the proliferation of BMSCs.
3.3 ABs from the middle stage of apoptosis are the most potent to induce migration of BMSCs
We used the scratch healing assay and Boyden chamber assay to compare the effect of ABs from different stages of apoptosis on the migration of BMSCs. Scratch healing assay showed that ABs accelerated the movement of m-BMSCs to the scratch area. Surprisingly, ABs from the middle stages of apoptosis had the highest ability to promote wound healing (Fig. 4C). Similar results that ABs from the middle stage of apoptosis have the highest ability to promote wound closure were found in r-BMSCs (Fig. 4D). The Boyden chamber assay showed that ABs accelerated the migration of m-BMSCs from the upper chamber to the lower chamber, but the number of migrating cells was highest in the STS-treated 4 h group (Fig. 4E). These results were consistent with the effect of ABs on r-BMSCs (Fig. 4F). Taken together, ABs promoted migration of BMSCs, and ABs from the middle stages of apoptosis had the strongest ability to promote migration.
3.4 ABs from the middle stage of apoptosis are the most potent to induce osteogenesis of BMSCs
To compare the osteogenic potency of ABs from different stages of apoptosis on BMSCs, m-BMSCs were co-cultured with ABs from different stages of apoptosis during osteogenic induction. Alizarin red staining (Fig. 5A-B) showed that ABs enhanced the capacities of m-BMSCs for osteogenic differentiation. Interestingly, ABs from the middle stage of apoptosis showed significantly higher osteogenic potency compared to other stages of apoptosis. We confirmed that the level of osteogenic Runx2 and ALP in recipient BMSCs were upregulated after exposure to ABs derived from the middle stage of apoptosis (Fig. 5C). We also confirmed this observation in r-BMSCs. The osteogenic differentiation assay with r-BMSCs showed that ABs enhanced the osteogenic differentiation of r-BMSCs. Treatment with ABs from the middle stage of apoptosis resulted in the highest degree of mineralization and the most of calcium nodules (Fig. 5D-E). Alizarin red staining and qRT-PCR (Fig. 5F) showed consistent results suggesting the highest osteogenic potency of r-BMSCs after co-cultured with ABs from the middle stage of apoptosis. Taken together, ABs can promote the osteogenic differentiation of BMSCs, and ABs from the middle stage of apoptosis have the highest ability to promote osteogenic differentiation.