The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist

Cristina Menchón; Michael J. Edel; Juan Carlos Izpisua Belmonte

doi:10.4161/cc.10.9.15421

The cell cycle inhibitor p27^Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist

Cristina Menchón, Michael J. Edel, Juan Carlos Izpisua Belmonte

Research output: Contribution to journal › Article › peer-review

37 Scopus citations

Abstract

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27^Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27^Kip1 in hESC lead to a G₁ phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27^Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27^Kip1 protein occupies the Twist1 gene promoter and manipulation of p27 ^Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27^Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27^Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.

Original language	English (US)
Pages (from-to)	1435-1447
Number of pages	13
Journal	Cell Cycle
Volume	10
Issue number	9
DOIs	https://doi.org/10.4161/cc.10.9.15421
State	Published - May 1 2011
Externally published	Yes

Bibliographical note

Funding Information:
with an annealing temperature of 58°C. PCPCR products were obtained after 30 cycle%R ps oProdf amOucpliPts wficUeatioEre n JTasThUsise auStaJnCce wthoVrs aUith floFw cytometry, Meritxell Carrió for expert re indebted to José Miguel Andrés Vaquero for cloned into pGEM-T easy vector (Promega Biotech Iberica) and assistance with cell culture techniques, Mercé Martí Gaudes, digested with Bam H1 and Asp 718 restriction enzymes. The Lola Mulero Pérez and Esther Melo for bioimaging assistance. fragment was cloned into Bam H1 and Asp 718 sites of p27 Dr. Naldini for providing pCCL vector. Federico González for shRNA and non target shRNA. helpful expertise with the cloning. Members of the laboratory Lentiviruses were independently produced by transfecting the for helpful discussion. María José Barrero for assistance with cell line 293T with packaging and envelope vectors: pMDLg-the ChIP assay. Cristina Menchón is partly supported by a pre-pRRE (6.5 μg), pMD2.VSV.G (4 μg), pRSV-Rev (2.5 μg) doctoral grant from the Spanish Ministry (AP-2004-3523). kindly provided by L. Naldini (San Raffaele Telethon Institute This work was partially supported by grants from the Fondo de for Gene Therapy-‘Vita-Salute San Raffaele’ University Medical Investigaciones Sanitarias (P1071209), MICINN, Fundacion School, Milano, Italy) and lentiviral constructs (10 μg) using Cellex and Sanofi-Aventis.

Keywords

Cell cycle
Differentiation
Embryonic stem cell
Induced pluripotent stem cell
Pluripotency
Self renewal

ASJC Scopus subject areas

Molecular Biology
Developmental Biology
Cell Biology

Access to Document

10.4161/cc.10.9.15421

Cite this

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title = "The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist",

abstract = "The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27Kip1 in hESC lead to a G1 phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27Kip1 protein occupies the Twist1 gene promoter and manipulation of p27 Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.",

keywords = "Cell cycle, Differentiation, Embryonic stem cell, Induced pluripotent stem cell, Pluripotency, Self renewal",

author = "Cristina Mench{\'o}n and Edel, {Michael J.} and Belmonte, {Juan Carlos Izpisua}",

note = "Funding Information: with an annealing temperature of 58°C. PCPCR products were obtained after 30 cycle%R ps oProdf amOucpliPts wficUeatioEre n JTasThUsise auStaJnCce wthoVrs aUith floFw cytometry, Meritxell Carri{\'o} for expert re indebted to Jos{\'e} Miguel Andr{\'e}s Vaquero for cloned into pGEM-T easy vector (Promega Biotech Iberica) and assistance with cell culture techniques, Merc{\'e} Mart{\'i} Gaudes, digested with Bam H1 and Asp 718 restriction enzymes. The Lola Mulero P{\'e}rez and Esther Melo for bioimaging assistance. fragment was cloned into Bam H1 and Asp 718 sites of p27 Dr. Naldini for providing pCCL vector. Federico Gonz{\'a}lez for shRNA and non target shRNA. helpful expertise with the cloning. Members of the laboratory Lentiviruses were independently produced by transfecting the for helpful discussion. Mar{\'i}a Jos{\'e} Barrero for assistance with cell line 293T with packaging and envelope vectors: pMDLg-the ChIP assay. Cristina Mench{\'o}n is partly supported by a pre-pRRE (6.5 μg), pMD2.VSV.G (4 μg), pRSV-Rev (2.5 μg) doctoral grant from the Spanish Ministry (AP-2004-3523). kindly provided by L. Naldini (San Raffaele Telethon Institute This work was partially supported by grants from the Fondo de for Gene Therapy-{\textquoteleft}Vita-Salute San Raffaele{\textquoteright} University Medical Investigaciones Sanitarias (P1071209), MICINN, Fundacion School, Milano, Italy) and lentiviral constructs (10 μg) using Cellex and Sanofi-Aventis.",

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month = may,

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doi = "10.4161/cc.10.9.15421",

language = "English (US)",

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AU - Edel, Michael J.

AU - Belmonte, Juan Carlos Izpisua

N1 - Funding Information: with an annealing temperature of 58°C. PCPCR products were obtained after 30 cycle%R ps oProdf amOucpliPts wficUeatioEre n JTasThUsise auStaJnCce wthoVrs aUith floFw cytometry, Meritxell Carrió for expert re indebted to José Miguel Andrés Vaquero for cloned into pGEM-T easy vector (Promega Biotech Iberica) and assistance with cell culture techniques, Mercé Martí Gaudes, digested with Bam H1 and Asp 718 restriction enzymes. The Lola Mulero Pérez and Esther Melo for bioimaging assistance. fragment was cloned into Bam H1 and Asp 718 sites of p27 Dr. Naldini for providing pCCL vector. Federico González for shRNA and non target shRNA. helpful expertise with the cloning. Members of the laboratory Lentiviruses were independently produced by transfecting the for helpful discussion. María José Barrero for assistance with cell line 293T with packaging and envelope vectors: pMDLg-the ChIP assay. Cristina Menchón is partly supported by a pre-pRRE (6.5 μg), pMD2.VSV.G (4 μg), pRSV-Rev (2.5 μg) doctoral grant from the Spanish Ministry (AP-2004-3523). kindly provided by L. Naldini (San Raffaele Telethon Institute This work was partially supported by grants from the Fondo de for Gene Therapy-‘Vita-Salute San Raffaele’ University Medical Investigaciones Sanitarias (P1071209), MICINN, Fundacion School, Milano, Italy) and lentiviral constructs (10 μg) using Cellex and Sanofi-Aventis.

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KW - Differentiation

KW - Embryonic stem cell

KW - Induced pluripotent stem cell

KW - Pluripotency

KW - Self renewal

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