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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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. 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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0269122</identifier><identifier>PMID: 36413521</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>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</subject><ispartof>PloS one, 2022-11, Vol.17 (11), p.e0269122</ispartof><rights>Copyright: © 2022 Nakanoh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</rights><rights>COPYRIGHT 2022 Public Library of Science</rights><rights>2022 Nakanoh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2022 Nakanoh et al 2022 Nakanoh et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c641t-76858813d1275cce8f4261e208380066394d35683ae533b6743a83b66ea70213</cites><orcidid>0000-0002-9016-7992</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9681086/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9681086/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2926,23865,27923,27924,53790,53792,79371,79372</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36413521$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Wu, Qiang</contributor><creatorcontrib>Nakanoh, Shota</creatorcontrib><creatorcontrib>Kadiwala, Juned</creatorcontrib><creatorcontrib>Pinte, Laetitia</creatorcontrib><creatorcontrib>Morell, Carola Maria</creatorcontrib><creatorcontrib>Lenaerts, An-Sofie</creatorcontrib><creatorcontrib>Vallier, Ludovic</creatorcontrib><title>Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells</title><title>PloS one</title><addtitle>PLoS One</addtitle><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.</description><subject>Analysis</subject><subject>Antibiotics</subject><subject>Biology and Life Sciences</subject><subject>Cell cycle</subject><subject>Cell differentiation</subject><subject>Cell Differentiation - physiology</subject><subject>Cell fate</subject><subject>Cell lines</subject><subject>Cell proliferation</subject><subject>Cell survival</subject><subject>CRISPR</subject><subject>Cyclin-dependent kinases</subject><subject>Depletion</subject><subject>Differentiation</subject><subject>Embryo cells</subject><subject>Embryonic stem cells</subject><subject>Endoderm</subject><subject>Endoderm - metabolism</subject><subject>Epigenetics</subject><subject>Foregut</subject><subject>Frameshift mutation</subject><subject>Gene expression</subject><subject>Growth</subject><subject>Human Embryonic Stem Cells - 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physiology</topic><topic>Cell fate</topic><topic>Cell lines</topic><topic>Cell proliferation</topic><topic>Cell survival</topic><topic>CRISPR</topic><topic>Cyclin-dependent kinases</topic><topic>Depletion</topic><topic>Differentiation</topic><topic>Embryo cells</topic><topic>Embryonic stem cells</topic><topic>Endoderm</topic><topic>Endoderm - metabolism</topic><topic>Epigenetics</topic><topic>Foregut</topic><topic>Frameshift mutation</topic><topic>Gene expression</topic><topic>Growth</topic><topic>Human Embryonic Stem Cells - metabolism</topic><topic>Humans</topic><topic>Kinases</topic><topic>Maintenance</topic><topic>Medicine and Health Sciences</topic><topic>Mutation</topic><topic>Physiological aspects</topic><topic>Pluripotency</topic><topic>Pluripotent Stem Cells - metabolism</topic><topic>Proteins</topic><topic>Redundancy</topic><topic>Research and Analysis Methods</topic><topic>Retinoblastoma Protein - genetics</topic><topic>Retinoblastoma-Like Protein p130 - genetics</topic><topic>Stem cells</topic><topic>Transcription factors</topic><topic>Tumor suppressor genes</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nakanoh, Shota</creatorcontrib><creatorcontrib>Kadiwala, Juned</creatorcontrib><creatorcontrib>Pinte, Laetitia</creatorcontrib><creatorcontrib>Morell, Carola Maria</creatorcontrib><creatorcontrib>Lenaerts, An-Sofie</creatorcontrib><creatorcontrib>Vallier, Ludovic</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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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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