Somatic cell reprogramming generates iPSCs in multiple pluripotency states, the two extremes being referred to as “naive” and “primed” [7;10]. Whereas naive iPSCs are successfully derived directly from murine somatic cells, human iPSCs tend to persist in a primed state and additional steps are required to achieve full conversion into naive iPSCs. Naive iPSCs closely resemble ESCs, exhibiting self-renewal capacity and developmental potential, poorly achieved by primed iPSCs. Hence, a priority of stem cell research is to generate naive iPSCs, which are thought to hold the best potential for therapeutic use.
Stage 1 (S1) intermediate colonies yielded a sufficient number of cells for functional assays and were independent of OKSM expression. Stage 2 (S2) nascent iPSCs were obtained after 4 additional days in culture, without doxycycline and the three compounds cocktail. Naive iPSCs were generated by prolonged culture in medium complemented with serum and Leukemia Inhibitory Factor (LIF), which prevents differentiation.
One advantage of this rapid reprogramming protocol is to reduce cellular heterogeneity due to the variability in reprogramming efficiency, the occurrence of molecular abnormalities during the conversion process [15;16], and the asynchrony resulting from the long culture periods required to reach the naive state . To further select homogenous populations of iPSCs, flow cytometry was extensively used in this study. For functional assays, isolation of iPSCs expressing fluorescent reporter genes was specifically performed using microfluidic On-Chip Sort technology, which is designed for the sterile handling of small size samples in culture medium, while minimizing physical damage associated with alternative methods [18;19]. This strategy allowed for best preservation of the iPSCs destined to be injected into 4n blastocyst to generate iPSCs-derived mice.
The higher developmental potential of S2 nascent iPSCs may be due to their greater ability to exit the pluripotency state and initiate differentiation. Indeed, the authors assessed the ability of S2 cells to differentiate and generate embryoid bodies (EB) upon removal of LIF from the culture medium. Interestingly, EB formed by S2 cells showed a greater down-regulation of pluripotency markers than naive iPSCs. These data suggested that S2 nascent iPSCs were more prone to differentiation than established naive iPSCs. This could be partly explained by the greater expression of primed pluripotency markers, such as the epiblast marker Otx2 , in S2 cells vs naive iPSCs. Indeed, after injection into 4n blastocysts, Oct4-GFP+ Otx2-RFP+ cells isolated from S2 colonies successfully gave rise to viable mice. By contrast, Oct4-GFP+Otx2-RFP+ naive iPSCs, which express lower level of Otx2, performed poorly in the 4n complementation assay.
1. Cossu G, Birchall M, Brown T, De Coppi P, Culme-Seymour E, Gibbon S, Hitchcock J, Mason C, Montgomery J, Morris S, Muntoni F, Napier D, Owji N, Prasad A, Round J, Saprai P, Stilgoe J, Thrasher A and Wilson J. (2018). Lancet Commission: Stem cells and regenerative Medicine. Lancet 391, 883-910.
2. Evans MJ and Kaufman MH. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156.
3. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998). Embryonic stem cell lines derived from human blastocysts. Science 282,1145-1147.
4. Takahashi K and Yamanaka S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 26, 663-676.
5. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872.
6. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II and Thomson JA. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318,1917-20.
7. Shi Y, Inoue H, Wu JC and Yamanaka S. (2017). Induced pluripotent stem cell technology: a decade of progress. Nat. Rev. Drug Discov. 16, 115-130.
8. Subramanian A, Sidhom EH, Emani M, Vernon K, Sahakian N, Zhou Y, Kost-Alimova M, Slyper M, Waldman J, Dionne D, Nguyen LT, Weins A, Marshall JL, Rosenblatt-Rosen O, Regev A and Greka A. (2019). Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation. Nat. Commun. 10, 5462.
9. Hunsberger JG, Rao M, Kurtzberg J, Bulte JWM, Atala A, LaFerla FM, Greely HT, Sawa A, Gandy S, Schneider LS, Doraiswamy PM. (2016). Accelerating stem cell trials for Alzheimer's disease. Lancet Neurol.15, 219-230.
10. Weinberger L, Ayyash M, Novershtern N and Hanna JH. (2016). Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Mol. Cell Biol. 17, 155-169.
11. Amlani, B, Liu Y, Chen T, Ee LS, Lopez P, Heguy A, Apostolou E, Kim SY and Stadtfeld M. (2018). Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes. Cell Reports 22, 876-884.
12. Stadtfeld M, Maherali N, Borkent M and Hochedlinger K. (2010). A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nat. Methods 7, 53-55.
13. Vidal SE, Amlani B, Chen T, Tsirigos A and Stadtfeld M. (2014). Combinatorial modulation of signaling pathways reveals cell-type-specific requirements for highly efficient and synchronous iPSC reprogramming. Stem Cell Reports 3, 574-584.
14. Stadtfeld M, Apostolou E, Ferrari F, Choi J, Walsh RM, Chen T, Ooi SS, Kim SY, Bestor TH, Shioda T, et al. (2012). Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nat. Genet. 44, 398-405.
15. Stadtfeld M, Apostolou E, Akutsu H, Fukuda A, Follett P, Natesan S, Kono T, Shioda T and Hochedlinger K (2010). Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465, 175–181.
16. Wu G, Lei L and Schöler HR. (2017). Totipotency in the mouse. J. Mol. Med. (Berl.) 95, 687-694.
17. Yamanaka S. (2009). Elite and stochastic models for induced pluripotent stem cell generation. Nature 460, 49-52.
18. Watanabe M, Serizawa M, Sawada T, Takeda K, Takahashi T, Yamamoto N, Koizumi F and Koh Y. (2014). A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood. J. Transl. Med. 12, 143.
19. Kasuga K, Katoh Y, Nagase K and Igarashi K. (2017). Microproteomics with microfluidic-based cell sorting: Application to 1000 and 100 immune cells. Proteomics 17(13-14), 1600420.
20. Collier AJ, Panula SP, Schell JP, Chovanec P, Plaza Reyes A, Petropoulos S, Corcoran AE, Walker R, Douagi I, Lanner F and Rugg-Gunn PJ. (2017). Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States. Cell Stem Cell 20, 874-890.e7.
21. Kallur T, Blomberg P, Stenfelt S, Tryggvason K and Hovatta O. (2017). Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell Banking. Methods Mol. Biol. 1590,11-16.
22. International Stem Cell Initiative. (2018). Assessment of established techniques to determine developmental and malignant potential of human pluripotent stem cells. Nat. Comnun. 9, 1925.