Sandhya Sriram1, Nam-Young Kang2,3, Subha Subramanian1, Tannistha Nandi4, Samydurai Sudhagar5, Qiaorui Xing6,7, Gerine Jin-Ling Tong8, Allen Kuan-Liang Chen8, Thekkeparambil Chandrabose Srijaya9, Patrick Tan5,10,11, Yuin-Han Loh6,12, Young-Tae Chang2,13,14,15, Shigeki Sugii16,17,18. 1. Fat Metabolism and Stem Cell Group, Singapore Bioimaging Consortium, A*STAR, 11 Biopolis Way, Singapore, 138667, Singapore. 2. Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, A*STAR, 11 Biopolis Way, Singapore, 138667, Singapore. 3. Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), Pohang, 37673, Republic of Korea. 4. Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, 60 Biopolis Street, Genome #02-01, Singapore, 138672, Singapore. 5. Genome Institute of Singapore, 60 Biopolis Street, Genome, #02-01, Singapore, 138672, Singapore. 6. Epigenetics and Cell Fates Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore, 138673, Singapore. 7. School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore. 8. Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore. 9. Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603, Kuala Lumpur, Malaysia. 10. Duke-NUS Medical School, 8 College Road, Singapore, 169857, Singapore. 11. SingHealth/Duke-NUS Institute of Precision Medicine, Singapore, 168752, Singapore. 12. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore. 13. Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore, 117543, Singapore. 14. Department of Chemistry, POSTECH, Pohang, Gyeongbuk, 37673, Republic of Korea. 15. Center for Self-assembly and Complexity, Institute for Basic Science (IBS), Pohang, 37673, Republic of Korea. 16. Fat Metabolism and Stem Cell Group, Singapore Bioimaging Consortium, A*STAR, 11 Biopolis Way, Singapore, 138667, Singapore. shigekis@ibn.a-star.edu.sg. 17. Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, 8 College Road, Singapore, 169857, Singapore. shigekis@ibn.a-star.edu.sg. 18. Institute of Bioengineering and Nanotechnology, A*STAR, 31 Biopolis Way, Singapore, 138669, Singapore. shigekis@ibn.a-star.edu.sg.
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.
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 humaniPS cells. METHODS: We screened the in-house built fluorescent library compounds that specifically bind humaniPS 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 humaniPS 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 humaniPS cells.
Entities:
Keywords:
Adipose-derived stromal cell (ASC); DOFLA library fluorescence dye; Dental pulp stem cell (DPSC); Early stage pluripotency; Golgi marker; Human induced pluripotent stem cell (hiPSC); Mesenchymal-epithelial transition (MET); Three-dimensional (3D) microcarrier-based culture system; Tra-1-60; cAMP responsive element binding protein (CREB)
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