Abstract
BackgroundDespite recent rapid progress in method development and biological understanding of induced pluripotent stem (iPS) cells, there has been a relative shortage of tools that monitor the early reprogramming process into human iPS cells.MethodsWe screened the in-house built fluorescent library compounds that specifically bind human iPS cells. After tertiary screening, the selected probe was analyzed for its ability to detect reprogramming cells in the time-dependent manner using high-content imaging analysis. The probe was compared with conventional dyes in different reprogramming methods, cell types, and cell culture conditions. Cell sorting was performed with the fluorescent probe to analyze the early reprogramming cells for their pluripotent characteristics and genome-wide gene expression signatures by RNA-seq. Finally, the candidate reprogramming factor identified was investigated for its ability to modulate reprogramming efficiency.ResultsWe identified a novel BODIPY-derived fluorescent probe, BDL-E5, which detects live human iPS cells at the early reprogramming stage. BDL-E5 can recognize authentic reprogramming cells around 7 days before iPS colonies are formed and stained positive with conventional pluripotent markers. Cell sorting of reprogrammed cells with BDL-E5 allowed generation of an increased number and higher quality of iPS cells. RNA sequencing analysis of BDL-E5-positive versus negative cells revealed early reprogramming patterns of gene expression, which notably included CREB1. Reprogramming efficiency was significantly increased by overexpression of CREB1 and decreased by knockdown of CREB1.ConclusionCollectively, BDL-E5 offers a valuable tool for delineating the early reprogramming pathway and clinically applicable commercial production of human iPS cells.
Highlights
Despite recent rapid progress in method development and biological understanding of induced pluripotent stem cells, there has been a relative shortage of tools that monitor the early reprogramming process into human iPS cells
We previously found that adipose-derived stem cells (ASCs) and dental pulpderived stem cells (DPSCs) allow feeder-free reprogramming with relatively high efficiencies and shorter time frames [4,5,6]
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, serving as stringent negative controls for authentic pluripotent stem cells
Summary
Despite recent rapid progress in method development and biological understanding of induced pluripotent stem (iPS) cells, there has been a relative shortage of tools that monitor the early reprogramming process into human iPS cells. Despite rapid development of methods to derive human iPS cells, there have been several problems and challenges with reprogramming protocols These include relatively low efficiency of obtaining high-quality cells, long duration of complete reprogramming processes (typically 3–4 weeks before colony formation), and difficulty in prompt analysis and identification of high quality iPS cells [2, 3]. Alkaline phosphatase and SSEA3/4 are not very specific to bona fide pluripotent stem cells [9] and reported to be detectable in adult stem cells including ASCs and DPSCs [10, 11] All of these markers typically stain well-developed colonies of iPS cells only, which can be visible and recognized by experienced observers even under phase contrast microscopy. We subsequently found that cAMP responsive element binding protein (CREB1) plays a role in the reprogramming process into human iPS cells
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