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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Veröffentlicht in:PloS one 2022-11, Vol.17 (11), p.e0269122
Hauptverfasser: Nakanoh, Shota, Kadiwala, Juned, Pinte, Laetitia, Morell, Carola Maria, Lenaerts, An-Sofie, Vallier, Ludovic
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container_title PloS one
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Kadiwala, Juned
Pinte, Laetitia
Morell, Carola Maria
Lenaerts, An-Sofie
Vallier, Ludovic
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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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. 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>36413521</pmid><doi>10.1371/journal.pone.0269122</doi><tpages>e0269122</tpages><orcidid>https://orcid.org/0000-0002-9016-7992</orcidid><oa>free_for_read</oa></addata></record>
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subjects Analysis
Antibiotics
Biology and Life Sciences
Cell cycle
Cell differentiation
Cell Differentiation - physiology
Cell fate
Cell lines
Cell proliferation
Cell survival
CRISPR
Cyclin-dependent kinases
Depletion
Differentiation
Embryo cells
Embryonic stem cells
Endoderm
Endoderm - metabolism
Epigenetics
Foregut
Frameshift mutation
Gene expression
Growth
Human Embryonic Stem Cells - metabolism
Humans
Kinases
Maintenance
Medicine and Health Sciences
Mutation
Physiological aspects
Pluripotency
Pluripotent Stem Cells - metabolism
Proteins
Redundancy
Research and Analysis Methods
Retinoblastoma Protein - genetics
Retinoblastoma-Like Protein p130 - genetics
Stem cells
Transcription factors
Tumor suppressor genes
title Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells
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