Opportunities and challenges for induced Pluripotent Stem Cells (iPSCs)

The new insights from a mouse model of rapidly reprogrammed nascent iPSCs

Written by Patricia Viard, PhD, HDR

Due to their ability to differentiate into nearly any cell type, pluripotent stem cells (PSCs) hold great promise for regenerative medicine ^[1], with the potential to treat numerous pathologies such as Parkinson and Alzheimer diseases, ischemic brain injury, spinal cord damage, heart and kidney failure, diabetes and macular degeneration.

Induced Pluripotent Stem Cells (iPSCs) vs Embryonic Stem Cells (ESCs)

Pluripotent cells were originally obtained from the inner cell mass of mouse ^[2] or human^[3] pre-implantation embryos (Embryonic Stem Cells: ESCs). However, the use of human ESCs is greatly restricted due to inherent ethical issues and the limited access to human embryos available for research. Over the past decade, an alternative approach was developed to derive pluripotent stem cells from somatic cells, through the forced expression of specific transcription factors, typically Oct4, Klf4, Sox2 and c-Myc (aka OKSM), to generate the so-called induced PSCs (iPSCs) in mouse ^[4] and human ^[5;6]. This ground-breaking approach was recognized by the award of the Nobel prize to Yamanaka in 2012. In addition to solving some of the ethical issues, this strategy offers a novel tool for disease modeling ^[7] and tissue-engineering ^[8], and paves the way for individualized cell-based therapies ^[9].

Naive and primed pluripotency states

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.

Superior developmental potential of nascent murine iPSCs

In a remarkable study published in Cell Reports in 2018, Amlani and colleagues ^[11] compared the developmental potential of intermediate (Stage 1), nascent (Stage 2) and established naive iPSCs derived from transgenic mouse embryonic fibroblasts (MEF).

Opportunities and challenges for induced Pluripotent Stem Cells (iPSCs)

Derivation of IPSCs from MEFs was achieved by inducing doxycycline-dependent expression of OKSM transgenes ^[12]. Rapid reprogramming was achieved by exposing MEFs to a combination of a GSK3 inhibitor, which activates Wnt signalling, with an antagonist of Tumor Growth Factor β receptor 1, and ascorbic acid, which promotes chromatin remodeling ^[13] and may contribute to the modification of epigenetic marks ^[14].

After 6 days of culture, 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 ^[17]. 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 main discovery from Amlani and colleagues ^[11] is that nascent S2 iPSCs, which are yet to acquire the hallmarks of the pluripotent naive state, exhibited greater developmental potential than established naive iPSCs in 4n complementation assays, and successfully generated fertile mice. These results indicate that developmental pluripotency is already determined in nascent iPSCs prior to the establishment of the naive pluripotency state. Interestingly, S2 cells were less able to self-renew than established naive iPSCs, indicating that self-renewal ability is not predictive of iPSCs developmental potential.

Opportunities and challenges for induced Pluripotent Stem Cells (iPSCs)

This study also shows that extensive chromatin remodeling, silencing of somatic genes, and upregulation of RNA transcripts associated with cell cycle progression and pluripotency (such as Oct4) have already occurred in S1 intermediate iPSCs. However, S1 cells showed no potential for development when injected into 4n blastocysts. To avoid any bias due to cellular heterogeneity, only S1 and S2 cells expressing the fluorescent reporter Oct4-GFP gene (Oct4-GFP+) were selected for injection into 4n blastocysts. In contrast to S2 cells, S1 iPSCs failed to generate viable mice, arguing that further key changes in e.g., the accessibility of genomic regulatory regions, or protein abundance, are required during the transition from S1 to S2 cells to promote development.

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 ^[10], 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.

Conclusions and perspectives

Nascent murine iPSCs provide a new paradigm for investigating the key molecular and cellular events involved in the emergence of the naive pluripotency state. In Human, recent advances have been made to identify cell surface markers characteristic of nascent iPSCs ^[20]. These could be used in a combined approach, using monoclonal antibodies and flow cytometry, to select the best performing iPSCs for regenerative therapies and disease modeling. Such strategy would also find application for the quality control of stem cells biobanking ^[21]. Most importantly, accurate and reliable selection of iPSCs is a pre-requisite for the safety of clinical trials, owing to the latent tumorigenicity of iPSCs that may fail to differentiate after grafting ^[22].

"Stay informed with the latest news from PHCbi”

Reference

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.

About PHCbi

The “bi” part of our new brand PHCbi is not only a shortened form of the word “biomedical” but also represents both our strength and philosophy as an abbreviation of “biomedical innovation.” Since the launch of our first Pharmaceutical Refrigerator model in 1966, we have taken advantage of this technology to create exceptional medical and laboratory products and services with a high degree of quality and reliability. We have worked to meet the expectations of customers in the medical and life science fields under both the Sanyo and Panasonic brands. See more detail from "About PHCbi". Find PHCbi's customer case study in the world.