PhD Student Center for iPS Cell Research and Application (CiRA), Kyoto University Kyoto, Kyoto, Japan
Abstract: Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells that can differentiate into osteocytes, chondrocytes, and adipocytes in vitro, and are being applied in several clinical treatments. MSCs can be differentiated from human induced pluripotent stem cells (iPSCs), and our group has developed a protocol to generate induced MSCs (iMSCs) from iPSCs via the neural crest cell (NCC) lineage. To maximize the value of iMSCs for medical applications, we aim to generate highly functional iMSCs and, to this end, employ a method to transfect functional genes into iPSCs and establish transgene-overexpressing iMSCs. However, transgene silencing may occur during iPSC differentiation, which poses a significant challenge to our research. Here, we explore methods to avoid silencing using cHS4 insulator sequences combined with the piggyBac transposon system. We tested a polycistronic vector expressing neutrophil elastase (ELANE), EGFP, and Puromycin-resistant genes under the regulation of CAG, EF1A, or CMV promoter. Our results suggest that the cHS4 insulator is effective with the CMV promoter, evidenced by the higher puromycin resistance of cHS4 (+) cells during NCC expansion. Although there is no clear difference between EF1A promoter with and without cHS4 in terms of ELANE and EGFP expressions during NCC and iMSC inductions, gene expression of ELANE and EGFP was decreased during inductions, suggesting that EF1A promoter is not suitable for our stable expression system. CAG promoter is most suitable for our system because iMSCs from CAG promoter with and without cHS4 expressed the highest levels of ELANE mRNA and protein, and their expressions are higher in cHS4 (+) cells compared with cHS4 (-) cells. Now we are trying more complex vectors and optimizing vector design to achieve a more efficient and robust expression system in iMSCs.
Funding Source: Management Expense Grant from University
