Abstract
Pluripotent stem cell-derived mesenchymal progenitor cells (PSC-MPCs) are primarily derived through two main methods: three-dimensional (3D) embryoid body-platform (EB formation) and the 2D direct differentiation method. We recently established somatic cell nuclear transfer (SCNT)-PSC lines and showed their stemness. In the present study, we produced SCNT-PSC-MPCs using a novel direct differentiation method, and the characteristics, gene expression, and genetic stability of these MPCs were compared with those derived through EB formation. The recovery and purification of SCNT-PSC-Direct-MPCs were significantly accelerated compared to those of the SCNT-PSC-EB-MPCs, but both types of MPCs expressed typical surface markers and exhibited similar proliferation and differentiation potentials. Additionally, the analysis of gene expression patterns using microarrays showed very similar patterns. Moreover, array CGH analysis showed that both SCNT-PSC-Direct-MPCs and SCNT-PSC-EB-MPCs exhibited no significant differences in copy number variation (CNV) or single-nucleotide polymorphism (SNP) frequency. These results indicate that SCNT-PSC-Direct-MPCs exhibited high genetic stability even after rapid differentiation into MPCs, and the rate at which directly derived MPCs reached a sufficient number was higher than that of MPCs derived through the EB method. Therefore, we suggest that the direct method of differentiating MPCs from SCNT-PSCs can improve the efficacy of SCNT-PSCs applied to allogeneic transplantation.
Highlights
CHA Advanced Research Institute, CHA University Bundang Medical Center, These authors contributed to this work
Only 1–2% of the single-nucleotide polymorphism (SNP) variations in all mesenchymal progenitor cells (MPCs) were detected during differentiation, and these variations did not differ by differentiation methods or time in culture in vitro until passage 12. These results indicate that there were no differences in the characteristics or proliferation capacity of somatic cell nuclear transfer (SCNT)-Pluripotent stem cells (PSCs)-MPCs and Embryonic stem cells (ESCs)-MPCs on the basis of their derivation by the EB or Direct method
We developed a direct differentiation method for MPCs derived from SCNT-PSCs and observed that their characteristics, such as morphology, expression level of cellular surface markers, and gene expression patterns, are very similar to those of ESC-MPCs derived through conventional differentiation methods via EB formation
Summary
CHA Advanced Research Institute, CHA University Bundang Medical Center, These authors contributed to this work. We produced SCNT-PSC-MPCs using a novel direct differentiation method, and the characteristics, gene expression, and genetic stability of these MPCs were compared with those derived through EB formation. Array CGH analysis showed that both SCNT-PSC-Direct-MPCs and SCNT-PSC-EB-MPCs exhibited no significant differences in copy number variation (CNV) or single-nucleotide polymorphism (SNP) frequency. These results indicate that SCNT-PSC-Direct-MPCs exhibited high genetic stability even after rapid differentiation into MPCs, and the rate at which directly derived MPCs reached a sufficient number was higher than that of MPCs derived through the EB method. Many reports have suggested that MPCs with low levels of HLA-DR expression and high levels of immune-modulating activity can be allogeneic grafts that cause no immune rejection [3,9]
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