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Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population
- Source :
- Stem Cell Research & Therapy, Vol 12, Iss 1, Pp 1-15 (2021), Stem Cell Research & Therapy
- Publication Year :
- 2021
- Publisher :
- BMC, 2021.
-
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.
- Subjects :
- 0301 basic medicine
Cell type
Induced Pluripotent Stem Cells
Cell
Population
Adipose-derived stromal cell (ASC)
Method
Medicine (miscellaneous)
Biology
Biochemistry, Genetics and Molecular Biology (miscellaneous)
lcsh:Biochemistry
03 medical and health sciences
0302 clinical medicine
Human induced pluripotent stem cell (hiPSC)
medicine
Dental pulp stem cell (DPSC)
Tra-1-60
Humans
lcsh:QD415-436
Induced pluripotent stem cell
education
cAMP responsive element binding protein (CREB)
Mesenchymal-epithelial transition (MET)
Cells, Cultured
Fluorescent Dyes
DOFLA library fluorescence dye
education.field_of_study
lcsh:R5-920
Three-dimensional (3D) microcarrier-based culture system
Golgi marker
Cell Biology
Cell sorting
Cellular Reprogramming
Cell biology
030104 developmental biology
medicine.anatomical_structure
Cell culture
Early stage pluripotency
Molecular Medicine
Stem cell
Transcriptome
lcsh:Medicine (General)
Reprogramming
030217 neurology & neurosurgery
Subjects
Details
- Language :
- English
- ISSN :
- 17576512
- Volume :
- 12
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Stem Cell Research & Therapy
- Accession number :
- edsair.doi.dedup.....6bd6ace34223d1a4769636e583eb81fb