Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells

Abstract

Abstract.The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm 2 . After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox (Nanog), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog (c-Myc), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc, were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent).These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

An Approach towards a GMP Compliant In-Vitro Expansion of Human Adipose Stem Cells for Autologous Therapies

“…However, after 10 days, the cells were hyperconfluent in both cases. Consequently, the expression of almost all genes decreased [42][43][44]. Nonetheless, the cells on the MCs performed well and their gene expression pattern was similar to that of those obtained from hASCs grown in static 2D conditions.…”

“…The differences in the gene expression profiles were more pronounced on day 5. However, on day 10, due to the very high cell density, most of the stemness genes decreased and most of the differentiation genes increased in both systems, resulting in similar profiles [42][43][44]. These results clearly demonstrated that for both donors, the optimum point of harvest was on day 5-6 (see Section 3.3).…”

“…Positive hASC markers (CD26 + , CD54 + , CD55 + , CD73 + and CD90 + ) were strongly expressed, while negative markers (CD36 − and CD146 − ) were only weakly expressed. CD105 was the only positive marker that was weakly expressed, which might be caused by the hyperconfluence of the cells after 10 days of cultivation [ 42 , 43 , 44 ].…”

“…As already mentioned, on day 10, the cells were hyperconfluent in both cases. In this situation it is normal that the stemness genes are downregulated, while the differentiation genes are induced [42,44]. However, it is worth noting that the expression of PPARγ, the master regulator of adipogenesis, was lower for both donors (080, 085) under dynamic 3D conditions.…”

The Influence of Cell Culture Density on the Cytotoxicity of Adipose-Derived Stem Cells Induced by L-Ascorbic Acid-2-Phosphate

“…A microarray study that systematically compared gene expression profiles of ASCs showed that proliferation-related genes were upregulated in cultures at ~50% confluence, whereas immunity and defense, cell communication, signal transduction, and cell motility genes were more highly expressed in cultures at ~90% confluence 30 . Moreover, stemness markers Nanog and c-Myc were altered in ASCs according to various seeding densities 31 . Sukho et al also reported that ASC sheets of a higher cellular density increased the expression levels of vascular endothelial growth factors and FGF2, whereas specific pro-inflammatory genes such as tumor necrosis factor-α and prostaglandin synthase 2 were downregulated 32 .…”