As the incidence of chronic kidney disease (CKD) increases, so does the need for novel therapeutics and cellular reprogramming may represent one such option. As all the nephrons in the human kidney arise prior to birth, regeneration of complete nephrons may require the recreation of embryonic nephron progenitors (NPs). We previously identified a set of 6 transcription factors (SIX1, SIX2, HOXA11, OSR1, EYA1 and SNAI2) that, when overexpressed using a lentivirus-mediated gene delivery system, were sufficient to reimpose a NP-like state in adult human proximal tubule cells (Hendry et al., JASN, 2013). Following on from this, we are currently investigating whether reprogramming can be achieved with fewer factors and if the efficiency and selectivity of this process can be improved.
In the first instance, here we report the preliminary de-replication of this gene set. Through sequentially removing individual lentiviral constructs from HK2 cell transductions, we found that a NP-like state could be achieved in the absence of EYA1 or HOXA11. Secondly, we have sought to improve this reprogramming process by developing a multicistronic construct, consisting of all 6 genes engineered into a piggyBac transposon with intervening 2A sequences, doxycycline inducibility and a fluorescent reporter (mCherry) for cell enrichment. Initial experiments utilizing this novel system indicated that transposon integration into HK2 cells is most efficient with the hyperactive piggyBac transposase (m7pB). Following transposon integration, HK2 cells displayed tightly regulated doxycycline-inducible mCherry expression. Increased expression of key NP markers was also detected, with the expression of these genes influenced by the length of doxycycline exposure.
These results further refine the genes required for NP reprogramming and demonstrate the feasibility of a transposon-based approach for inducible reprogramming to a NP. In the long term, this approach will facilitate patient-specific reprogramming to NP cells. Such cells may also prove invaluable for bioengineering and nephrotoxicity screening.