High-throughput tracking of pluripotent human embryonic stem cells with dual fluorescence resonance energy transfer molecular beacons.

Stem Cells and Development
Frank W KingHarold S Bernstein

Abstract

Pluripotent human embryonic stem cells (hESCs) provide an unprecedented opportunity for the study of human tissue development, and the development of cell-based therapies for human disease. To realize these potential advances, however, methods for monitoring expression of intracellular proteins in live hESCs without altering cellular properties are needed. Molecular beacons are single-stranded oligonucleotides that have been employed to assay gene expression. To test their potential for high-throughput isolation of hESCs, we developed a dual fluorescence resonance energy transfer (FRET) molecular beacon system using fluorescence-activated cell sorting (FACS) with Oct4 as a target. We demonstrate that Oct4 can be detected by FRET using confocal microscopy, that this can be applied in a high-throughput manner to the identification and isolation of Oct4-expressing hESCs by FACS, that FRET-positive hESCs demonstrate pluripotency in culture and in vivo, and that hESCs transfected with molecular beacons demonstrate normal growth rates and oligonucleotide extinction over time. These studies demonstrate that FRET-based FACS using molecular beacons provides a useful tool for isolating Oct4-expressing pluripotent hESCs, and may also be a...Continue Reading

References

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Citations

Oct 31, 2012·Science China. Life Sciences·Sheng TongGang Bao
Mar 21, 2012·Pediatric Research·Harold S Bernstein, Deepak Srivastava
May 12, 2012·Stem Cell Research & Therapy·Harold S Bernstein, William C Hyun
Dec 5, 2012·PloS One·Hans M LarssonJeffrey A Hubbell
Sep 8, 2012·Regenerative Medicine·Harold S Bernstein
Jul 15, 2015·Expert Opinion on Biological Therapy·Bernat SoriaAbdelkrim Hmadcha
Mar 5, 2016·Nature Communications·Allison J CoteRobert L Mauck
Dec 23, 2014·The Analyst·Hisakage FunabashiAkio Kuroda
Oct 8, 2016·Cellular and Molecular Life Sciences : CMLS·Parvin Abraham, Tessy Thomas Maliekal
Mar 15, 2015·Stem Cells Translational Medicine·Yuanjian GuoSuzanne Scarlata
Mar 9, 2019·Tissue Engineering. Part a·Yuki MurataYasuhiko Tabata
Jul 5, 2019·Advanced Materials·Devleena SamantaChad A Mirkin
Mar 2, 2017·Cellular and Molecular Life Sciences : CMLS·Ho-Chang JeongHyuk-Jin Cha

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Methods Mentioned

BETA
fluorescence-activated cell sorting
FRET
reverse
transfection
flow cytometry
fluorescence resonance
confocal microscopy
FACS
PCR

Software Mentioned

RNAfold
FlowJo
MetaMorph
NIH Image
StereoVision

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