Therapeutic Potential of Small Extracellular Vesicles Derived from Lipoma Tissue in Adipose Tissue Regeneration – An in Vitro and in Vivo Study
Authors: Hong Pengyu , Xu Xiaoyang , Hu Xin , Yang Hao , Wu Yue , Chen Juan , Li Kun , Tang Zhangui 
Published: January 2021

Abstract
Objective
To explore the adipogenic effects of the small extracellular vesicles derived from the lipoma tissues (sEV-LT), and to find a new cell-free therapeutic approach for adipose tissue regeneration.
Methods
Adipose tissue-derived stem cells (ADSCs) and small extracellular vesicles derived from the adipose tissues (sEV-AT) were isolated from human adipose tissue, while sEV-LT were isolated from human lipomatous tissue. ADSCs were characterized by using flow cytometric analysis, adipogenic and osteogenic differentiation assays. sEV was identified by electron microscopy, nanoparticle tracking and western blotting. ADSCs were treated with sEV-LT and sEV-AT, respectively. Fluorescence confocal microscopy were used to investigate whether sEV-LT and sEV-AT could be taken by ADSCs. The proliferation, migration and adipogenic differentiation of ADSCs were compared by CCK-8 assays, scratch test and oil red O staining test, and the expression levels of adipogenic-related genes C/EBP-δ, PPARγ2 and Adiponectin in ADSCs were compared by real-time quantitative PCR (RT-PCR). The sEV-LT and sEV-AT transplantation tubes were implanted subcutaneously in SD rats, and the neotissues were qualitatively and histologically evaluated in 2, 4, 8 and 12 weeks after transplantation. Hematoxylin and eosin (H&E) staining was used to observe and compare the adipogenesis and angiogenesis in neotissues, while immunohistochemistry was used to examine the expression and distribution of C/EBP-α, PPARγ, Adiponectin and CD31 at the 4th week.
Results
Both sEV-LT and sEV-AT could be taken up by ADSCs via endocytosis in vitro experiments. The scratch experiment and CCK-8 experiment showed that the migration area and proliferation number of ADSCs in sEV-LT group and sEV-AT group were significantly higher than those in the non-sEVs group(p < 0.05). Compared with sEV-AT group, sEV-LT group had larger migration area and proliferation number of ADSCs(p < 0.05). Oil red O staining and RT-PCR experiments showed that, compared with the group without sEVs, the lipid droplets and the mRNA expression levels of adipogenesis-related genes PPARγ2 and Adiponectin of ADSCs in sEV-LT group and sEV-AT group were significantly up-regulated(p < 0.05), while the expression level of C/EBP-δ was not statistically significant compared to the group without sEVs (p > 0.05); Compared with sEV-AT groups, ADSCs in sEV-LT groups showed no statistically significant difference in the amount of lipid droplets and adipogenesis-related genes(p > 0.05). At 2, 4, 8 and 12 weeks, the adipocyte area and the number of capillaries in neotissues in the sEV-LT groups and sEV-AT groups were significantly increased compared with the Matrigel group(p < 0.05); Compared with sEV-AT groups, sEV-LT groups showed no significant difference in adipocyte area and the number of capillaries in neotissues(p > 0.05). At the 4th week, neotissues in the sEV-LT groups and sEV-AT groups all showed positive expression of C/EBP-α, PPARγ, Adiponectin and CD31 protein, while neotissues in the Matrigel group only showed positive expression of CD31 protein.
Conclusions
This study demonstrated that sEV-LT exerted promotion effects on adipose tissue regeneration by accelerating the proliferation and migration and adipogenic differentiation of ADSCs in vitro, recruiting adipocytes and promoting angiogenesis in vivo. sEV-LT could serve as an alternative cell-free therapeutic strategy for generating adipose tissue, thus providing a promising application prospect in tissue engineering.