One major challenge in stem cell research is to identify and sort the various subpopulations of cells co-existing within heterogeneous cultures. Indeed, stem cells (SCs), obtained either directly from the inner cell mass of preimplantation embryos (ESCs) [1;2] or by reprogramming somatic cells (induced Pluripotent SCs/iPSCs) [3;4;5], exist in two main pluripotency states, either primed or naive, with a range of intermediate states[6;7]. In addition to their greater self-renewal abilities, naive SCs have a greater capacity to differentiate into cell-types of interest when receiving appropriate cues [7;8]. Then again, there is a strong variability within the cells regarding the efficiency and timing of the differentiation process. Only a fraction of SCs reach the desired phenotype and can be used for cell-based therapy or pharmaceutical studies . Furthermore, SCs which fail to differentiate are potentially teratogenic and accurate cell selection is primordial for the safety of clinical applications . Hence, reliable techniques are needed to isolate naive SCs and, after in vitro differentiation, SC-derived cells of interest.
Seminal studies in SC selection were performed using transgenic animal models expressing fluorescent reporter genes [3;7;11]. However, there are inherent biases associated with the use of transgenes. Their expression may interfere with the normal function of the cell by competing with endogenous proteins, preventing interactions with partners or regulators. In addition, random insertion of transgenes into the genomic DNA may alter endogenous gene regulation and/or integrity. Alternatively, immunodetection of cell surface markers has been used to achieve cell type selection in live conditions [12;13;14]. However, immunolabeling is not readily reversible. Hence, none of these techniques are suitable for human therapy. Thus, there is a growing interest for non-invasive techniques to detect endogenous cell markers of either pluripotency or differentiation. A range of fluorescent probes have been developed to select either pluripotent SCs or differentiated cells. Remarkable examples are described below.
1. Evans MJ and Kaufman MH. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156.
2. 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.
3. Takahashi K, Yamanaka S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 26, 663-676.
4. 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.
5. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318,1917-20.
6. Weinberger L, Ayyash M, Novershtern N, Hanna JH. (2016). Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Mol. Cell Biol. 17, 155-169.
7. Shi Y, Inoue H, Wu JC, Yamanaka S. (2017). Induced pluripotent stem cell technology: a decade of progress. Nat. Rev. Drug Discov. 16, 115-130.
8. Ware CB, Nelson AM, Mecham B, Hesson J, Zhou W, Jonlin EC, Jimenez-Caliani AJ, Deng X, Cavanaugh C, Cook S, Tesar PJ, Okada J, Margaretha L, Sperber H, Choi M, Blau CA, Treuting PM, Hawkins RD, Cirulli V, Ruohola-Baker H. (2014). Derivation of naïve human embryonic stem cells. Proc Natl Acad Sci U S A. 111, 4484-9.
9. 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 ,Wilson J. (2018). Lancet Commission: Stem cells and regenerative Medicine. Lancet 391, 883-910.
10. Sato Y, Bando H, Di Piazza M, Gowing G, Herberts C, Jackman S, Leoni G, Libertini S, MacLachlan T, McBlane JW, Pereira Mouriès L, Sharpe M, Shingleton W, Surmacz-Cordle B, Yamamoto K, van der Laan JW. (2019). Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider. Cytotherapy 21, 1095-1111.
11. Amlani B, Liu Y, Chen T, Ee LS, Lopez P, Heguy A, Apostolou E, Kim SY, Stadtfeld M. (2018). Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes. Cell Rep.22, 876-884.
12. Boheler KR, Bhattacharya S, Kropp EM, Chuppa S, Riordon DR, Bausch-Fluck D, Burridge PW, Wu JC, Wersto RP, Chan GC, Rao S, Wollscheid B, Gundry RL. (2014). A human pluripotent stem cell surface N-glycoproteome resource reveals markers, extracellular epitopes, and drug targets. Stem Cell Reports 3,185-203.
13. Collier AJ, Panula SP, Schell JP, Chovanec P, Plaza Reyes A, Petropoulos S, Corcoran AE, Walker R, Douagi I, Lanner F, 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.
14. Goodwin J, Laslett AL, Rugg-Gunn PJ. (2020). The application of cell surface markers to demarcate distinct human pluripotent states. Exp. Cell Res. 387(1), 111749.
15. Yun SW, Kang NY, Park SJ, Ha HH, Kim YK, Lee JS, Chang YT (2014). Diversity oriented fluorescence library approach (DOFLA) for live cell imaging probe development. Acc Chem Res. 47, 1277-1286.
16. Im CN, Kang NY, Ha HH, Bi X, Lee JJ, Park SJ, Lee SY, Vendrell M, Kim YK, Lee JS, Li J, Ahn YH, Feng B, Ng HH, Yun SW, Chang YT (2010). A fluorescent rosamine compound selectively stains pluripotent stem cells. Angew Chem Int Ed Engl.49, 7497-4500.
17. Kang N, Yun S, Ha H, Park SJ, Chang YT. (2011) Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1. Nat Protoc 6, 1044–1052.
18. Cho SJ, Kim SY, Park SJ, Song N, Kwon HY, Kang NY, Moon SH, Chang YT, Cha HJ. (2016). Photodynamic Approach for Teratoma-Free Pluripotent Stem Cell Therapy Using CDy1 and Visible Light. ACS Cent Sci. 2, 604-607.
19. Lee AS, Tang C, Rao MS, Weissman IL, Wu JC. (2013). Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies, Nat. Med. 19, 998-1004.
20. Hirata N, Nakagawa M, Fujibayashi Y, Yamauchi K, Murata A, Minami I, Tomioka M, Kondo T, Kuo TF, Endo H, Inoue H, Sato SI, Ando S, Kawazoe Y, Aiba K, Nagata K, Kawase E, Chang YT, Suemori H, Eto K, Nakauchi H, Yamanaka S, Nakatsuji N, Ueda K, Uesugi M. (2014). A chemical probe that labels human pluripotent stem cells. Cell Rep. 6, 1165-1174.
21. Chandran Y, Kang NY, Park SJ, Husen Alamudi S, Kim JY, Sahu S, Su D, Lee J, Vendrell M, Chang YT. (2015). A highly selective fluorescent probe for direct detection and isolation of mouse embryonic stem cells. Bioorg Med Chem Lett. 25, 4862-4865.
22. Cho SJ, Kim KT, Kim JS, Kwon OS, Go YH, Kang NY, Heo H, Kim MR, Han DW, Moon SH, Chang YT, Cha HJ. (2018). A fluorescent chemical probe CDy9 selectively stains and enables the isolation of live naïve mouse embryonic stem cells. Biomaterials.180,12-23.
23. Berk PD, Stremmel W. (1986). Hepatocellular uptake of organic anions. Prog Liver Dis.8:125-44.
24. Yamada T, Yoshikawa M, Kanda S, Kato Y, Nakajima Y, Ishizaka S, Tsunoda Y. (2002). In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green. Stem Cells 20, 146-154.
25. Yoshie S, Ito J, Shirasawa S, Yokoyama T, Fujimura Y, Takeda K, Mizuguchi M, Matsumoto K, Tomotsune D, Sasaki K. (2012). Establishment of novel detection system for embryonic stem cell-derived hepatocyte-like cells based on nongenetic manipulation with indocyanine green. Tissue Eng Part C Methods 18,12-20.
26. Yun SW, Leong C, Zhai D, Tan YL, Lim L, Bi X, Lee JJ, Kim HJ, Kang NY, Ng SH, Stanton LW, Chang YT (2012). Neural stem cell specific fluorescent chemical probe binding to FABP7. Proc Natl Acad Sci U S A. 109, 10214-10217.
27. Rhee WJ, Bao G. (2009). Simultaneous detection of mRNA and protein stem cell markers in live cells. BMC Biotechnol. 9, 30.
28. Ban K, Wile B, Cho KW, Kim S, Song MK, Kim SY, Singer J, Syed A, Yu SP, Wagner M, Bao G, Yoon YS. (2015). Non-genetic Purification of Ventricular Cardiomyocytes from Differentiating Embryonic Stem Cells through Molecular Beacons Targeting IRX-4. Stem Cell Reports 5, 1239-1249.
29. Lahm H, Doppler S, Dreßen M, Werner A, Adamczyk K, Schrambke D, Brade T, Laugwitz KL, Deutsch MA, Schiemann M, Lange R, Moretti A, Krane M. (2015). Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species. Stem Cells. 33, 392-402.