Research Assistant K.S. Hegde Medical Academy, India
Abstract: Adipose-derived mesenchymal stem cells (ASCs) offer an ideal source for cell-based regenerative therapies due to their easy accessibility and multipotency features. The present study evaluated the impact of in vitro expansion on the morphology, growth, presence of surface markers, lineage differentiation and the expression of molecular markers related to immunomodulation, proliferation, senescence, and aging of clinical-grade human ASCs obtained from early (2-3) and late (9-10) passages. The adherent cells of ASCs cultured in the serum-free media (SFM) exhibited a fibroblast-like morphology. However, the cells with robust proliferative ability were more abundant in cultures of early passages. Cells expressed the mesenchymal markers (CD44, CD73, CD90, CD105) but not the hematopoietic markers (CD34, CD45, HLA-DR), and no difference in expression was observed between the early and late passages. But, late ASCs displayed an enlarged senescent-like morphology, and reduced clonogenic capacity when compared to early ASCs. Upon induction, ASCs were differentiated into osteogenic, adipocytic and chondrogenic lineages. However, early ASCs had higher propensity towards the formation of osteocytes, adipocytes and chondrocytes as evidenced by cytochemical staining and the expression of lineage specific molecular markers. The expression levels of markers assessed by ELISA and qPCR related to immunomodulation, such as interleukin (IL)-1 beta, IL-6, TNF-alpha (pro-inflammatory cytokines), IL-10, TGF-beta and IL-12 (anti-inflammatory cytokines) showed variations with the duration of the ASCs expansion. The markers pertaining to the proliferation (SIRT1 and Ki67) were downregulated in late ASCs. In contrast, the molecular markers implicated in senescence and aging (p53, p21 and p16) showed the higher levels of expression in late ASCs. In conclusion, there was no significant difference in the expression of phenotypic markers among the passages of clinical-grade ASCs. However, the biological and molecular characteristics of ASCs cultured in SFM varied as the number of passages increased. Therefore, further optimization and assessment of cell performance are required in large scale culture system before using clinical-grade ASCs of late passages for therapeutic applications.
