INTRODUCTION: Autologous cell delivery to the herniated or degenerated intervertebral disc (IVD) may promote tissue regeneration and arrest degeneration. However, few sources of "healthy" autologous cells have been identified for IVD. Induced pluripotent stem cells (iPSCs) derived from the patient's somatic cells, with the potential of differentiating into various cell types represent an attractive cell source. To date, no studies have demonstrated that iPSCs can differentiate into nucleus pulposus (NP) cells. We have demonstrated that human umbilical cord mesenchymal stromal cells could differentiate into NP‐like cells in a laminin‐rich pseudo‐3D culture system. The goal of this study is to evaluate if mouse iPSCs cultured in this same laminin‐rich environment can express a unique NP‐like cell phenotype.
METHODS: iPSCs were generated from mouse embryonic fibroblasts through transient inducible over‐expression of transcription factors (OCT4, SOX2, KLF4 and MYC). Undifferentiated cells were formed into 3D embryoid bodies (EBs) in iPSC medium for 3 days, seeded (106/well, Transwell™) on wells pre‐coated with Matrigel, then cultured in differentiation media (DMEM/F12+10% FBS) containing 2.5% Matrigel to provide for a pseudo‐3D culture system. Cell surface marker and laminin expression were evaluated via flow cytometry, RT‐PCR and immunohistochemistry in cells at three different stages (undifferentiated, EBs, day 7 post‐NP differentiation).
RESULTS: Undifferentiated iPSCs had positive expression for many integrins (α3, α5, α6 and β1 subunit). Interestingly, expression of CD24, a NP cell marker, increased in EBs and differentiated cells as compared to undifferentiated cells. The expression of other NP markers (cytokeratin 8, type II collagen, aggrecan, laminin10/11 and its receptors) was also confirmed in differentiated cells. In conclusion, mouse iPSCs have the potential to express NP‐like phenotypes in a laminin‐rich pseudo‐3D culture environment.