Cargando…
Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population
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 lib...
Autores principales: | , , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866770/ https://www.ncbi.nlm.nih.gov/pubmed/33546754 http://dx.doi.org/10.1186/s13287-021-02171-6 |
_version_ | 1783648151049076736 |
---|---|
author | Sriram, Sandhya Kang, Nam-Young Subramanian, Subha Nandi, Tannistha Sudhagar, Samydurai Xing, Qiaorui Tong, Gerine Jin-Ling Chen, Allen Kuan-Liang Srijaya, Thekkeparambil Chandrabose Tan, Patrick Loh, Yuin-Han Chang, Young-Tae Sugii, Shigeki |
author_facet | Sriram, Sandhya Kang, Nam-Young Subramanian, Subha Nandi, Tannistha Sudhagar, Samydurai Xing, Qiaorui Tong, Gerine Jin-Ling Chen, Allen Kuan-Liang Srijaya, Thekkeparambil Chandrabose Tan, Patrick Loh, Yuin-Han Chang, Young-Tae Sugii, Shigeki |
author_sort | Sriram, Sandhya |
collection | PubMed |
description | 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13287-021-02171-6. |
format | Online Article Text |
id | pubmed-7866770 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-78667702021-02-08 Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population Sriram, Sandhya Kang, Nam-Young Subramanian, Subha Nandi, Tannistha Sudhagar, Samydurai Xing, Qiaorui Tong, Gerine Jin-Ling Chen, Allen Kuan-Liang Srijaya, Thekkeparambil Chandrabose Tan, Patrick Loh, Yuin-Han Chang, Young-Tae Sugii, Shigeki Stem Cell Res Ther Method 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13287-021-02171-6. BioMed Central 2021-02-05 /pmc/articles/PMC7866770/ /pubmed/33546754 http://dx.doi.org/10.1186/s13287-021-02171-6 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Method Sriram, Sandhya Kang, Nam-Young Subramanian, Subha Nandi, Tannistha Sudhagar, Samydurai Xing, Qiaorui Tong, Gerine Jin-Ling Chen, Allen Kuan-Liang Srijaya, Thekkeparambil Chandrabose Tan, Patrick Loh, Yuin-Han Chang, Young-Tae Sugii, Shigeki Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population |
title | Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population |
title_full | Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population |
title_fullStr | Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population |
title_full_unstemmed | Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population |
title_short | Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population |
title_sort | novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866770/ https://www.ncbi.nlm.nih.gov/pubmed/33546754 http://dx.doi.org/10.1186/s13287-021-02171-6 |
work_keys_str_mv | AT sriramsandhya novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT kangnamyoung novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT subramaniansubha novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT nanditannistha novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT sudhagarsamydurai novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT xingqiaorui novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT tonggerinejinling novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT chenallenkuanliang novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT srijayathekkeparambilchandrabose novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT tanpatrick novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT lohyuinhan novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT changyoungtae novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation AT sugiishigeki novellivecellfluorescentprobeforhumaninducedpluripotentstemcellshighlightsearlyreprogrammingpopulation |