ALP as a reliable marker for predicting early reprogramming

Objectives: Plasmid-based transfection by Yamanaka’s factors (OSKM) is thought to be more difficult to obtain iPS cells than the other viral transfection. However, repeated transfection can improve the above plasmid-based transfection for iPS cell generation. Previously, we found that cells with higher levels of alkaline phosphatase (ALP) activity are more efficiently induced to generate iPS cells than those with lower levels of ALP activity, when human deciduous dental pulp cells (HDDPCs) are subjected to single transfection with OSKM. In this study, we examined whether cells with higher ALP activity are susceptible to iPS cell induction through repeated plasmid-based transfection using cells with high ALP activity (hereafter referred to as “ALP-H”) and those with low ALP activity (hereafter referred to as “ALP-L”).
Methods: Cells were transfected twice (at an interval of 4 days) by plasmids carrying OSKM using Neon transfection system. Cytochemical staining for ALP activity was performed on 0, 2, 4, 6 and 8 days after the final transfection. RT-PCR analysis was performed using total RNA isolated from HDDPCs 8 days after transfection to examine the expression of stem cell markers. Finally, we measured the number of emerging iPS cell colonies to examine correlation between the efficiency of iPS cell establishment and elevated levels of ALP activity in HDDPCs.
Results: ALP activity in both type of cells was increased after repeated transfection. Particularly, ALP-H exhibited more increased ALP activity than ALP-L. Increased expression of ALP, OCT4, NANOG and KLF4 was more evident in ALP-H than in ALP-L. The number of colonies emerged is much higher in ALP-H than in ALP-L.
Conclusions: HDDPCs with higher ALP activity are more easily reprogrammed by OSKM. ALP activity may be one of the reliable marker for reporting early stage of reprogramming.