Ow it through the process of reprogramming. In this report, we demonstrate that pluripotent stem cells with the epiblast-like/primed state exhibit a characteristic blue fluorescence in standard media that arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The fluorescence is very easily detected working with wide field epifluorescence microscopy. It enables for effective single cell separation employing FACS and propagation. The fluorescence also serves as an early reprogramming marker for induced human pluripotent stem cells (HiPSCs). Finally, we show that whereas mouse embryonic stem cells (ESCs) don’t have fluorescent lipid bodies, they are present in pluripotent mouse epiblast-like cells (mEpiSCs) and in the epiblast area on the mouse embryo.RESULTSHuman Pluripotent Stem Cells Have Characteristic Blue Fluorescent Cytoplasmic Lipid Bodies HPSC cultures on mouse embryonic fibroblast (MEF) feeders in common media with serum or serum replacement exhibited a blue fluorescence easily observed by epifluorescence microscopy (excitation 32575 nm, emission 45000 nm) and readily captured having a cooled charge-coupled device camera (Figure 1A). The blue fluorescence was related with most cells inside colonies with typical human ESC (HuESC) colony morphology, although person cells had varied levels of fluorescence (Figure 1A). At higher magnification, the blue fluorescence wasStem Cell Reports j Vol.IL-1 beta Protein, Mouse 3 j 16984 j July eight, 2014 j 014 The AuthorsStem Cell ReportsRetinoid Fluorescence in Pluripotent Stem CellsFigure 1.Anetumab Human Pluripotent Stem Cells Have Cytoplasmic Lipid Bodies that Exhibit Characteristic Blue Fluorescence (A) Blue fluorescence (excitation, 325375 nm; emission, 46000 nm) was observed in HPSC (HuESC and HiPS; i.e., NFF_iPS, ADF_iPS, and LCL_iPS) colonies cultured in common media and culture circumstances. (B) Representative high-magnification confocal image of HuES7 cells showing blue fluorescence confined to spherical bodies. (C) The fluorescent spherical bodies typically show polarized distribution within cells (red arrows, upper row) and stain positive for lipid body-specific markers (BODIPY and Nile red; middle row). The merged images of BODIPY-blue fluorescence and Nile red-blue fluorescence are shown in the reduced row. BF, bright field; NFF, neonatal foreskin fibroblast; ADF, adult dermal fibroblast; LCL, lymphoblastoid cell line. See also Figure S1.connected with several spherical cytoplasmic bodies that had been 0.five mm (Figure 1B) and usually perinuclear (Figure 1C, red arrows). The fluorescence was retained on fixation with paraformaldehyde and prone to bleaching but recovered in reside cells (Figure 1C).PMID:23310954 The fluorescence is unlikely to be autofluorescence from dying cells for the reason that we do not see any autofluorescence at green or red wavelengths (Figure S1C out there on the web). These bodies were stained with lipid body-specific markers BODIPY and Nile red (Figure 1C) and were not related with other cytoplasmic compartments (Figure S1D). Human neonatal foreskin fibroblasts (NFF), MEFs, mesenchymal stem cells, and HPSC-derived neurons had considerably reduce blue fluorescence (Figures S1A and S1B). Blue Fluorescent Lipid Bodies Are Connected with Markers of Human Pluripotent Stem Cells HPSC colonies usually show signs of differentiation in culture apparent as regions of altered morphology. Pluripotency markers for instance OCT4, SOX2, and NANOG may be used to identify the differentiation status of HPSCs but these demand cells to be fixed a.