13. A Novel Method of Culturing Murine Induced Pluripotent Stem Cells (iPSCs)

Abstract

Similar to embryonic stem cells (ESCs), iPSCs have the ability to differentiate into all three cell lineages, while not being subjected to the ethical complications attributed to ESCs. Our project goal was to develop a novel method of culturing murine iPSCs, reprogrammed from mouse embryonic fibroblasts, in large-scale quantities while maintaining their pluripotent characteristics and genomic integrity over a long term period. Stirred Suspension Bioreactors (SSBs) propose several benefits over static culture systems and facilitate the large-scale, economical expansion required for clinical studies. In our work with the SSB system, various techniques were used to promote and analyze the continued pluripotency of the murine iPSCs. Our results showed that iPSCs maintained their pluripotent state in SSBs and retained their ability to differentiate into different cell lineages after long term culture. miPSC were induced to differentiate into heart, bone and cartilage tissues at the end of maintenance period. The cumulative cell-fold expansion of iPSCs in suspension culture was 8.8×1010 cells, with 75% mean pluripotency at each passage. The cells expressed the major pluripotency markers as detected by RT-PCR. This study confirms the utility of the SSB system as a tool to cultivate large quantities of functional mouse iPSCs. SSBs have tremendous potential in future clinical applications and work has now begun to applying this system towards culturing human iPSCs. One potential application is the derivation of disease and patient derived iPSCs, which can recapitulate the disease phenotype for use as an in vitro model.