Finally, we asked whether we could rescue the poor blood-forming potential of NP-iPSC by differentiation into hematopoietic lineages1633, followed by a tertiary round of reprogramming back to pluripotency by doxycycline induction of the endogenous reprogramming factors (Fig. 4a, lower schema). As a control, we differentiated NP-iPSCs into neural stem cells8, followed by tertiary reprogramming to pluripotency. Resulting iPSC clones were selected for expression of the Nanog-eGFP reporter and shown to express Oct4 and Nanog by immunohistochemistry (Supplementary Fig. 8) and to chimerize murine blastocysts (Supplementary Fig. 9). The tertiary blood-derived B-NP-iPSC showed higher hematopoietic colony-forming potential than the tertiary NSC-NP-iPSC (Fig. 4b), and generated larger hematopoietic colonies with more cells per colony (Supplementary Fig. 10a, b). These data indicate that the poor blood-forming potential of secondary NP-iPSC can be enhanced by differentiation into hematopoietic progeny, followed by tertiary reprogramming. In contrast, tertiary reprogramming via neural intermediates yields iPSC that retain poor hematopoietic potential.
