Supplementary MaterialsAdditional file 1
Supplementary MaterialsAdditional file 1. compounds for various cell selective imaging probe development, which was named Diversity Orientated Fluorescence Library Approach (DOFLA) . A high-throughput system using the in-house DOFLA [15C17] was employed to screen 46 fluorescent probes that we predicted may specifically recognize pluripotent stem cells. Twenty-nine out of the 46 probes were previously used for screening of AX20017 mouse ES cells [12, 13]. In this study, we expanded the library, which contains novel compounds mainly harboring rosamine and BODIPY derivatives. To identify fluorescent probes that detected human pluripotent stem cells, ASCs, ASC-derived iPS (AiPS) cells, DPSCs, and DPSC-derived iPS (DiPS) cells were AX20017 used. ASCs and DPSCs were chosen as these cells show relatively high reprogramming efficiencies and were previously shown to exhibit many of the conventional pluripotent markers, thus serving as stringent negative controls for authentic pluripotent stem cells. Cells were seeded onto 384-well plates (primary screen) or 96-well plates (secondary and tertiary screen) coated with mouse embryonic fibroblasts (MEF) or matrigel (MG) for DOFLA screening (Fig.?1a). After 48?h, cells were stained with library probes (500?nM) for 1?h. Fluorescence was imaged using the ImageXpress System under the following conditions: no wash (after 1?h probe incubation), wash 0 min (after one wash with PBS), wash 60 min (after wash and destain for 1?h), and wash 180 min (after wash and destain for 3?h) (Fig. ?(Fig.11a). Open in a separate window Fig. 1 a Schematic diagram showing the screening process of fluorescent probes for somatic and iPS cells using DOFLA. Cells were seeded on either MEFs or MG in 384-well plates for primary screening and 96-well plates for secondary and tertiary screening. After 48?h, cells were stained with probes for 1?h and images were taken in the ImageXpress under various conditions as indicated. b The chemical structure of the probe, BDL-E5, used in this study Images were analyzed using the MetaXpress Image Acquisition and Analysis software. Following tertiary screening, two probes were shortlisted to develop further as pluripotent probes: BDL-E5 and CDy1. CDy1 is among 29 probes that AX20017 were previously screened against mouse ES cells and was extensively studied [12, 13]. The chemical structure of BDL-E5 is depicted in Fig. ?Fig.1b.1b. BDL-E5 is based on 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), with calculated mass of 528.3 and absorption maximum/emission maximum of 578/599?nm. These two probes showed significantly increased intensity of fluorescence in human iPS cells compared with their original somatic cells and MEFs. Figure?2a shows increased BDL-E5 staining (no wash) in AiPS colonies grown on MEF- or MG-coated plates, compared with their original somatic cells from the primary screening. Figure S1 shows increased CDy1 staining in AiPS cells on MEF- or MG-coated plates compared with ASCs from the primary screen. Open in a separate window Fig. 2 a Fluorescent images (10X objective) of BDL-E5 probe (no wash) and Hoechst 33342 staining of AiPS1 colonies and its original ASC1 (ASC line #1) on MEF- (i) and MG-coated (ii) plates from primary screening (and (Fig.?5eCh). BDL-E5+ cells showed decreased expression of compared with BDL-E5? cells. BDL-E5? cells AX20017 showed increased expression of mesenchymal genes comparable to DPSCs. Gene expression measurements using qPCR in reprogrammed ASCs sorted with BDL-E5 at 14 dpn showed increased and expression in BDL-E5+ cells, while IL7 BDL-E5? cells showed significantly increased expression of and slightly increased expression of (a); (b); (c); and (d); (e); (f), (g); and (h) in RNA isolated from DiPS2, DPSC2, BDL-E5+, and BDL-E5? cells of DPSC2 obtained after FACS at 7 dpn (and (Fig. S4B(ii)C(v)). Hence, these results supported authenticity of the BDL-E5+-derived iPS cells. BDL-E5 detects iPS cells generated with common protocols or three-dimensional (3D) culture conditions and may localize to the Golgi complex In order to test whether BDL-E5 would be useful for identifying reprogramming and iPS cells in 3D culture suitable for large scale production, ASCs and DPSCs were reprogrammed and seeded on Geltrex-coated Cytodex 3 microcarriers prior to staining with BDL-E5 and TRA-1-60. As shown in Fig. S4C, BDL-E5 staining was clearly observed in reprogrammed cells in both cell lines, and pluripotency of the iPS cells formed on the microcarriers was confirmed using TRA-1-60.