Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population

Abstract

Background 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. Methods We 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. Results We 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. Conclusion Collectively, BDL-E5 offers a valuable tool for delineating the early reprogramming pathway and clinically applicable commercial production of human iPS cells.

Culturing and Scaling up Stem Cells of Dental Pulp Origin Using Microcarriers

“…To date, only three studies have reported DPSC cultivation on microspheres in dynamic, three-dimensional shaking conditions [38,40,[60][61][62]. These three papers used dynamic conditions but were concerned only with cell differentiation and gene expressional changes and lacked any quantitative cell viability or cell proliferation data [40,61,62]. Cell expansion of DPSCs on microcarriers has been characterized in only two studies, using stationary and not dynamic conditions, as described above [38,60].…”

CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics

“…Attempts to resolve these pertinent issues surrounding both iPSCs and CRISPR/Cas9 technology are currently under active exploration. For example, highly precise surface-marker and fluorescence-based cell sorting approaches to yield pure populations of iPSCs and iPSC-derivatives have been reported by several groups (Lehnen et al 2017 ; Arias-Fuenzalida et al 2017 ; Schmitt et al 2017 ; Hoshina et al 2018 ; Patterson et al 2020 ; Sriram et al 2021 ). Several recent reports have also described promising approaches to improving the efficiency and reliability of the CRISPR/Cas9 system while minimizing the occurrences of unwanted off-target edits.…”