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Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells

RB is a well-known cell cycle regulator controlling the G1 checkpoint. Previous reports have suggested that it can influence cell fate decisions not only by regulating cell proliferation and survival but also by interacting with transcription factors and epigenetic modifiers. However, the functional...

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Autores principales: Nakanoh, Shota, Kadiwala, Juned, Pinte, Laetitia, Morell, Carola Maria, Lenaerts, An-Sofie, Vallier, Ludovic
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9681086/
https://www.ncbi.nlm.nih.gov/pubmed/36413521
http://dx.doi.org/10.1371/journal.pone.0269122
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author Nakanoh, Shota
Kadiwala, Juned
Pinte, Laetitia
Morell, Carola Maria
Lenaerts, An-Sofie
Vallier, Ludovic
author_facet Nakanoh, Shota
Kadiwala, Juned
Pinte, Laetitia
Morell, Carola Maria
Lenaerts, An-Sofie
Vallier, Ludovic
author_sort Nakanoh, Shota
collection PubMed
description RB is a well-known cell cycle regulator controlling the G1 checkpoint. Previous reports have suggested that it can influence cell fate decisions not only by regulating cell proliferation and survival but also by interacting with transcription factors and epigenetic modifiers. However, the functional redundancy of RB family proteins (RB, RBL1 and RBL2) renders it difficult to investigate their roles during early development, especially in human. Here, we address this problem by generating human embryonic stem cells lacking RB family proteins. To achieve this goal, we first introduced frameshift mutations in RBL1 and RBL2 genes using the CRISPR/Cas9 technology, and then integrated the shRNA-expression cassette to knockdown RB upon tetracycline treatment. The resulting RBL1/2_dKO+RB_iKD cells remain pluripotent and efficiently differentiate into the primary germ layers in vitro even in the absence of the RB family proteins. In contrast, we observed that subsequent differentiation into foregut endoderm was impaired without the expression of RB, RBL1 and RBL2. Thus, it is suggested that RB proteins are dispensable for the maintenance and acquisition of cell identities during early development, but they are essential to generate advanced derivatives after the formation of primary germ layers. These results also indicate that our RBL1/2_dKO+RB_iKD cell lines are useful to depict the detailed molecular roles of RB family proteins in the maintenance and generation of various cell types accessible from human pluripotent stem cells.
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spelling pubmed-96810862022-11-23 Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells Nakanoh, Shota Kadiwala, Juned Pinte, Laetitia Morell, Carola Maria Lenaerts, An-Sofie Vallier, Ludovic PLoS One Research Article RB is a well-known cell cycle regulator controlling the G1 checkpoint. Previous reports have suggested that it can influence cell fate decisions not only by regulating cell proliferation and survival but also by interacting with transcription factors and epigenetic modifiers. However, the functional redundancy of RB family proteins (RB, RBL1 and RBL2) renders it difficult to investigate their roles during early development, especially in human. Here, we address this problem by generating human embryonic stem cells lacking RB family proteins. To achieve this goal, we first introduced frameshift mutations in RBL1 and RBL2 genes using the CRISPR/Cas9 technology, and then integrated the shRNA-expression cassette to knockdown RB upon tetracycline treatment. The resulting RBL1/2_dKO+RB_iKD cells remain pluripotent and efficiently differentiate into the primary germ layers in vitro even in the absence of the RB family proteins. In contrast, we observed that subsequent differentiation into foregut endoderm was impaired without the expression of RB, RBL1 and RBL2. Thus, it is suggested that RB proteins are dispensable for the maintenance and acquisition of cell identities during early development, but they are essential to generate advanced derivatives after the formation of primary germ layers. These results also indicate that our RBL1/2_dKO+RB_iKD cell lines are useful to depict the detailed molecular roles of RB family proteins in the maintenance and generation of various cell types accessible from human pluripotent stem cells. Public Library of Science 2022-11-22 /pmc/articles/PMC9681086/ /pubmed/36413521 http://dx.doi.org/10.1371/journal.pone.0269122 Text en © 2022 Nakanoh et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Nakanoh, Shota
Kadiwala, Juned
Pinte, Laetitia
Morell, Carola Maria
Lenaerts, An-Sofie
Vallier, Ludovic
Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells
title Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells
title_full Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells
title_fullStr Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells
title_full_unstemmed Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells
title_short Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells
title_sort simultaneous depletion of rb, rbl1 and rbl2 affects endoderm differentiation of human embryonic stem cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9681086/
https://www.ncbi.nlm.nih.gov/pubmed/36413521
http://dx.doi.org/10.1371/journal.pone.0269122
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