Sorting Live Stem Cells Based on Sox2 MRNA Expression

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2012 Dec 5

PMID 23209609

Citations 16

Authors

Hans M Larsson

Seung Tae Lee

Marta Roccio

Diana Velluto

Matthias P Lutolf

Peter Frey

Jeffrey A Hubbell

Affiliations

Soon will be listed here.

Abstract

While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

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References

Boyer L, Mathur D, Jaenisch R . Molecular control of pluripotency. Curr Opin Genet Dev. 2006; 16(5):455-62. DOI: 10.1016/j.gde.2006.08.009. View

Tyagi S, Kramer F . Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996; 14(3):303-8. DOI: 10.1038/nbt0396-303. View

Okumura-Nakanishi S, Saito M, Niwa H, Ishikawa F . Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J Biol Chem. 2004; 280(7):5307-17. DOI: 10.1074/jbc.M410015200. View

Nitin N, Santangelo P, Kim G, Nie S, Bao G . Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells. Nucleic Acids Res. 2004; 32(6):e58. PMC: 390380. DOI: 10.1093/nar/gnh063. View

Zuker M . Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003; 31(13):3406-15. PMC: 169194. DOI: 10.1093/nar/gkg595. View

Rhee W, Bao G . Simultaneous detection of mRNA and protein stem cell markers in live cells. BMC Biotechnol. 2009; 9:30. PMC: 2682800. DOI: 10.1186/1472-6750-9-30. View

Graham V, Khudyakov J, Ellis P, Pevny L . SOX2 functions to maintain neural progenitor identity. Neuron. 2003; 39(5):749-65. DOI: 10.1016/s0896-6273(03)00497-5. View

Viswanathan S, Benatar T, Mileikovsky M, Lauffenburger D, Nagy A, Zandstra P . Supplementation-dependent differences in the rates of embryonic stem cell self-renewal, differentiation, and apoptosis. Biotechnol Bioeng. 2003; 84(5):505-17. DOI: 10.1002/bit.10799. View

Rhee W, Santangelo P, Jo H, Bao G . Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons. Nucleic Acids Res. 2008; 36(5):e30. PMC: 2275124. DOI: 10.1093/nar/gkn039. View

10.

Ellis P, Fagan B, Magness S, Hutton S, Taranova O, Hayashi S . SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev Neurosci. 2005; 26(2-4):148-65. DOI: 10.1159/000082134. View

11.

King F, Liszewski W, Ritner C, Bernstein H . High-throughput tracking of pluripotent human embryonic stem cells with dual fluorescence resonance energy transfer molecular beacons. Stem Cells Dev. 2010; 20(3):475-84. PMC: 3005532. DOI: 10.1089/scd.2010.0219. View

12.

Takahashi K, Yamanaka S . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-76. DOI: 10.1016/j.cell.2006.07.024. View

13.

Giachino C, Basak O, Taylor V . Isolation and manipulation of mammalian neural stem cells in vitro. Methods Mol Biol. 2008; 482:143-58. DOI: 10.1007/978-1-59745-060-7_9. View

14.

Basak O, Taylor V . Identification of self-replicating multipotent progenitors in the embryonic nervous system by high Notch activity and Hes5 expression. Eur J Neurosci. 2007; 25(4):1006-22. DOI: 10.1111/j.1460-9568.2007.05370.x. View

15.

Velluto D, Thomas S, Simeoni E, Swartz M, Hubbell J . PEG-b-PPS-b-PEI micelles and PEG-b-PPS/PEG-b-PPS-b-PEI mixed micelles as non-viral vectors for plasmid DNA: tumor immunotoxicity in B16F10 melanoma. Biomaterials. 2011; 32(36):9839-47. DOI: 10.1016/j.biomaterials.2011.08.079. View

16.

Mathews D, Sabina J, Zuker M, Turner D . Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol. 1999; 288(5):911-40. DOI: 10.1006/jmbi.1999.2700. View

17.

Pastrana E, Silva-Vargas V, Doetsch F . Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell. 2011; 8(5):486-98. PMC: 3633588. DOI: 10.1016/j.stem.2011.04.007. View

18.

Chambers I, Silva J, Colby D, Nichols J, Nijmeijer B, Robertson M . Nanog safeguards pluripotency and mediates germline development. Nature. 2007; 450(7173):1230-4. DOI: 10.1038/nature06403. View

19.

Lee S, Yun J, Jo Y, Mochizuki M, van der Vlies A, Kontos S . Engineering integrin signaling for promoting embryonic stem cell self-renewal in a precisely defined niche. Biomaterials. 2009; 31(6):1219-26. DOI: 10.1016/j.biomaterials.2009.10.054. View