|Differentiation Efficiency of Induced Pluripotent Stem Cells Depends on the Number of Reprogramming Factors|
From Stem Cells
Multiple studies in the last few years have attempted the production of induced pluripotent stem cells (iPSCs) with a reduced set of reprogramming factors (from OCT4, SOX2, KLF4, MYC or OCT4, SOX2, LIN28 and NANOG) in order that the process becomes streamlined in the hope that this may reduce potential mutation load in the resultant cells. This has been accomplished by many groups, resulting in iPSC formation at a lower efficiency, but the effect of a reduction in reprogramming factors on the subsequent differentiation capacity has not been fully explored. A report in the March edition of Stem Cells, from the laboratory of Alexander Storch now addresses this point, and finds that a reduction of reprogramming factors not only reduces reprogramming efficiency but negatively affects differentiation (Löhle et al).
Initial analysis of the loss of pluripotency during PA6 stromal cell co-culture mediated neuronal differentiation of embryonic stem cells (ESCs) and iPSCs derived from mouse embryonic fibroblasts using 4 factors (Oct4, Klf4, Sox2, c-Myc) (iPSC-MEF-4F) from Oct4-GFP transgenic mice found similar proportions of colonies with remaining Oct4-GFP+ throughout the 13 day protocol (between 60 and 40%). However, iPSCs derived from neural stem cells (NSCs) using 4 factors (iPSC-NSC-4F) lost most Oct4-GFP by 5 days of differentiation and decreased to 0% by day 13. Interestingly, prolonged Oct4-GFP expression was however observed in iPSCs derived from NSCs using either 1 factor (Oct4) or two factors (Oct4 and Klf4) (iPSC-NSC-1F and iPSC-NSC-2F). Subsequent analysis of neuronal markers found that Nestin, βIII-Tubulin, Map2 and Th expression demonstrated similar proportions of positively stained colonies in ESCs and iPSC-NSC-4F, while iPSC-NSC-1F and iPSC-NSC-2F produced fewer positively stained colonies. Further, in line with current thinking regarding epigenetic memory of origin dictating a preferred differentiation propensity, the iPSC-MEF-4F also demonstrated fewer Nestin+ colonies.
Functional analysis of cells after 4 weeks of differentiation demonstrated that Na+ channel voltage dependence and kinetics were reduced in the reduced factor iPSCs compared to the ESCs and 4 factor iPSC-NSC-4F cells, suggesting reduced functionality, while there was evidence of voltage-gated potassium channels in all cells. Further electrophysiological analysis suggested that the iPSC-NSC-4F derived cells had a maturational advantage over the reduced factor cells and only iPSC generated with four reprogramming factors showed additional spontaneous firing of action potentials at resting membrane conditions. Further functional analysis through the assessment of behavioural and histological outcome in 6-hydroxydopanime lesioned rats before and after transplantation of ESC and iPSC-NSC-2F differentiated for 11 days, found that iPSC-NSC-2F derived cells gave no significant improvement compared to ESC-derived cells, while histological examination found TH+ fibres and cell bodies after 9 weeks after ESC-derived cell transplant but only a few TH+ fibres in the iPSC-NSC-2F-derived cell implants and no TH+ cell bodies. Lastly, differentiation outwith the neuronal lineage was also assessed, and it was demonstrated that after hematoendothelial differentiation, reduced Pecam1+ or Flk1+ colonies were found for the iPSC-NSC-2F cells compared to ESCs and iPSC-MEF-4F and iPSC-NSC-4F cells.
Overall this suggests that while reducing the number of reprogramming factors may seem like an attractive process, the subsequent differentiation capacity of the cells may be altered insofar as making them unsuitable for regenerative medicine.
Löhle M et al.