Qualifying mscs for clinical applications -differential gene expression analyses of pluripotent, differentiation and surface marker genes of early and late passage mscs, in hypoxic and normal oxygen level in culture conditions.

Abstract

Background & Aim With the increasing focus on the large scale expansion of MSCs required for the clinical application, the optimisation of in-vitro expansion of MSCs requires careful consideration to maintain the native MSCs characteristics. Physiological parameters such as oxygen concentration, media constituents and passage numbers have much influence on the properties of MSCs and may have major impact on the therapeutic potential of MSCs. Cells grown under hypoxic (low oxygen) conditions have been widely documented in clinical use however culturing MSCS in large scale requires bioreactor and it is challenging to maintain the low oxygen and other physiological parameters over several passages in big vessels of bioreactors. Similarly, the culture conditions in the bioreactor including pH, dissolved oxygen and the sheer force exerted on cells may change the cellular characteristics including gene expression Methods, Results & Conclusion These culture conditions may have different potency attributes clinically. For these reasons, our study investigated differential expression of 11 key genes of MSCs for pluripotent genes POU5F1, NANOG and KLF4, differentiation genes RUNX2, COL1A1, SOX9, COL2A1, PPARG and CD marker genes THY1, ENG and NT5E from adipose derived MSCs, grown under both the normal oxygen or hypoxic conditions were analysed. Two different cells age early (P5) and late passage (P9) cells. The result demonstrated marked differences in gene expression pattern of the early and late passage cells grown under normoxic (normal oxygen) when compared with the hypoxic conditions. Pluripotent markers KLF4 in early passage cells was expressed more than two-fold in the normoxic cells, and PPARG was expressed more in the late passage cells under normoxic condition. Differentiation marker for RUNX2 and NT5E were highly expressed in early passage cells. These differences in gene expression may have critical impact on the potency of the cells grown in large scale in bioreactor. Additionally, the effects of prolonged culture of MSCs in-vitro will need be qualified prior to use when manufactured in large scale bioreactor for the clinical dose to ensure the therapeutic efficacy.