AL
reprogramming factors. Doxycycline was added from day 2 on, and the cells were seeded onto a murine embryonic fibroblast feeder layer on day 3. By day 9, colonies had formed, and probes specific for either NANOG or GDF3 were added. On day 10, individual colonies with either bright or faint fluorescence were identified under the microscope, replated on day 11 and expanded. After the second passage of these cells, pluripotency gene expression was evaluated by qRT-PCR.
The data demonstrated that NANOG is a reliable marker for the identi- fication of truly reprogrammed iPS cells. This conclusion was confirmed upon further evaluation in downstream differentiation experiments using murine embryonic stem cells as a control.3
NANOG-selected colonies ex-
pressed markers specific for endoderm, ectoderm or cardiac mesoderm at levels comparable to that of differentiated murine embryonic stem cells. Additionally, these clones allowed the identification of cardiac progeni- tors based on their green fluorescent protein (GFP) fluorescence. Colonies with a high NANOG-specific fluorescence generated a strongly increased fraction of cardiac progenitors upon differentiation. Therefore, NANOG- specific SmartFlare probes can be used to identify truly reprogrammed developing murine iPS cell colonies live and in situ.
Conclusion SmartFlare RNA detection probes can be used in a variety of cell types to reliably detect the expression of mRNA and miRNA in single live cells without manipulation of the target cell. The probes are nontoxic, do not affect mRNA or protein expression and do not impact cell proliferation. Importantly, the same treated cells can be used to study functionality in downstream experiments. This technology can be particularly powerful for studying heterogeneous cell populations and cell differentiation over time, identifying reprogrammed iPS cells and for functional assessment of RNA biomarkers.
References 1. Seferos, D.S.; Giljohann. D.A. et al. Nano-flares: probes for transfection and mRNA detection in living cells. J. Am. Chem. Soc. 2007, 129(50), 15,477–9.
2. Lahm, H.; Doppler, S. et al. Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent cells of different species. Stem Cells 2015, 33(2), 392–402; doi: 10.1002/ stem.1872.
3. Wu, S.M.; Fujiwara, Y. et al. Developmental origin of a bipotential myo- cardial and smooth muscle cell precursor in the mammalian heart. Cell 2006 Dec 15, 127(6), 1137–50.
Harald Lahm, Ph.D., is Head of Laboratory of Molecular and Cell Biol- ogy of Experimental Surgery, German Heart Center Munich. Don Weldon is R&D Manager, MilliporeSigma, 290 Concord Rd., Billerica, Mass. 01821, U.S.A.; tel.: 951-514-4566; e-mail:
don.weldon@emdmillipore.com; www.
emdmillipore.com. This article is based on Ref. 2 and a GenomeWeb webinar presented by Harald Lahm and Don Weldon.
AMERICAN LABORATORY 35
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