Gene expression profiling and genetic stability of human somatic cell nuclear transfer embryonic stem cell-derived mesenchymal progenitor cells produced by two different protocols

Abstract

Background & Aim As therapeutic cells, ES-MPC has been studied as a good alternative source to overcoming the disadvantages of adult MSC such as a limiting proliferative capacity and different biological characteristics result from various donor. There are various methods of producing ES-MPC, but can be divided into two main ways: the 3D platform (EB formation) method and the 2D culture (Direct) method. All of the ES-MPC differentiated using each method is known to have MSC-specific characteristics and tri-lineage differentiation capacities. However, there are still differences in proliferative capacity, therapeutic efficacy, differentiation duration and cost of ES-MPC according to differentiation methods. We developed a simple, direct differentiation method of hES-MPC. In this study, we produced ES-EB-MPC and ES-Direct-MPC from the same SCNT-hESC, and analyzed their characters, gene expression profiles, and genetic stability in order to understand ES-MPC characteristics and find the best protocol. Methods, Results & Conclusion We used CHA-hES NT17 and NT18 for this study. For Direct method, SCNT-hESC were treated with SB431542 for 3 days, and then additionally treated with RS-1 and Y27632, simultaneously. After 24 hour, the cells were transferred with collagenase and cultured. We have got homogenous cell population by serial sub-passaging. For EB method, SCNT-hESC were differentiated as described in our previous report (Cell Prolif. 2019). The ES-Direct-MPC and ES-EB-MPC were expressed by typical MSC markers and undergone tri-lineage differentiation similar to each other. Although ES-Direct-MPC were established faster than ES-EB-MPC, the analysis of gene expression patterns using microarray analysis between ES-Direct-MPC and ES-EB-MPC showed very similar patterns (scatter plot, R=0.99). Moreover, gene ontology analysis showed the same gene expression pattern classified by cellular function. ArrayCGH results showed that both ES-Direct-MPC and ES-EB-MPC had high genetic stability. Based on these results, there are no differences in MSC characteristics and proliferation capacity between EB and Direct methods, if so, the direct differentiation method we developed can save time and cost to produce ES-MPC. This research was supported by grants (No.2017M3A9C6061284, 2017M3A9F8072235 and 2019R1A6A1A03032888) from the Bio & Medical Technology Development Program of the NRF funded by the Korean government (MSIP).