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Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27
BACKGROUND: Adult stem cells exist in a quiescent state (G0) within the in vivo niche; the loss of quiescence often leads to a decrease in the number and function of adult stem cells, impairing tissue regeneration and repair. Endogenous repair by nucleus pulposus-derived stem cells has recently show...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466800/ https://www.ncbi.nlm.nih.gov/pubmed/30987681 http://dx.doi.org/10.1186/s13287-019-1219-8 |
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author | Li, Bin Sun, Chao Sun, Jing Yang, Ming-hui Zuo, Rui Liu, Chang Lan, Wei-ren Liu, Ming-han Huang, Bo Zhou, Yue |
author_facet | Li, Bin Sun, Chao Sun, Jing Yang, Ming-hui Zuo, Rui Liu, Chang Lan, Wei-ren Liu, Ming-han Huang, Bo Zhou, Yue |
author_sort | Li, Bin |
collection | PubMed |
description | BACKGROUND: Adult stem cells exist in a quiescent state (G0) within the in vivo niche; the loss of quiescence often leads to a decrease in the number and function of adult stem cells, impairing tissue regeneration and repair. Endogenous repair by nucleus pulposus-derived stem cells has recently shown promising regenerative potential for the treatment of intervertebral disc degeneration (IDD). However, the number and function of nucleus pulposus stem cells (NPSCs) declined throughout the process of IDD. This effect may have a specific relationship with quiescence. However, the biology of the quiescent NPSCs has not been reported. METHODS: First, we established an in vitro model for NPSC quiescence with serum starvation. The induction of G0 was confirmed by flow cytometry analyses of dual staining with Hoechst 33342 and Pyronin Y, immunofluorescent staining with Ki67 and Western blot analysis of P27 expression. NPSCs were cultured under serum starvation conditions for a long time period (21 days). To examine the functional phenotype of quiescent NPSCs, the cells were reactivated with 10% serum and differentiated into osteogenic and chondrogenic lineages in vitro. The number of colony-forming units was also estimated. To elucidate the role of autophagy in the quiescence of NPSCs, we activated and inhibited autophagy in starved cells with rapamycin and chloroquine, respectively. Then, the expression of P27 was evaluated by Western blot analysis, and the immunofluorescence of Ki67 was assessed. Finally, we assessed the role of P27 siRNA in NPSC quiescence by flow cytometry analyses and 5-ethynyl-20-deoxyuridine incorporation assays under normal and serum-starved conditions. RESULTS: NPSC quiescence was induced by 48 h of serum starvation, and they maintained quiescence for up to 21 days. Upon reactivation with serum, the quiescent NPSCs re-entered the cell cycle and exhibited enhanced clonogenic self-renewal, osteogenic differentiation and chondrogenic differentiation potentials compared to control NPSCs under normal culture conditions. We also found that autophagy underlay serum starvation-induced NPSC quiescence. Further study demonstrated that autophagy mediated the quiescence of NPSCs by regulating P27. CONCLUSIONS: Serum starvation efficiently induces quiescence in NPSCs. Quiescent NPSCs maintain stem cell properties. Our study reveals that autophagy plays a role in maintaining NPSC quiescence and that autophagy mediates the quiescence of NPSCs by regulating P27. We conclude that the induction of quiescence in cultured NPSCs provides a useful model for the analysis of mechanisms that might be relevant to the biology of NPSCs in vivo. |
format | Online Article Text |
id | pubmed-6466800 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-64668002019-04-22 Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27 Li, Bin Sun, Chao Sun, Jing Yang, Ming-hui Zuo, Rui Liu, Chang Lan, Wei-ren Liu, Ming-han Huang, Bo Zhou, Yue Stem Cell Res Ther Research BACKGROUND: Adult stem cells exist in a quiescent state (G0) within the in vivo niche; the loss of quiescence often leads to a decrease in the number and function of adult stem cells, impairing tissue regeneration and repair. Endogenous repair by nucleus pulposus-derived stem cells has recently shown promising regenerative potential for the treatment of intervertebral disc degeneration (IDD). However, the number and function of nucleus pulposus stem cells (NPSCs) declined throughout the process of IDD. This effect may have a specific relationship with quiescence. However, the biology of the quiescent NPSCs has not been reported. METHODS: First, we established an in vitro model for NPSC quiescence with serum starvation. The induction of G0 was confirmed by flow cytometry analyses of dual staining with Hoechst 33342 and Pyronin Y, immunofluorescent staining with Ki67 and Western blot analysis of P27 expression. NPSCs were cultured under serum starvation conditions for a long time period (21 days). To examine the functional phenotype of quiescent NPSCs, the cells were reactivated with 10% serum and differentiated into osteogenic and chondrogenic lineages in vitro. The number of colony-forming units was also estimated. To elucidate the role of autophagy in the quiescence of NPSCs, we activated and inhibited autophagy in starved cells with rapamycin and chloroquine, respectively. Then, the expression of P27 was evaluated by Western blot analysis, and the immunofluorescence of Ki67 was assessed. Finally, we assessed the role of P27 siRNA in NPSC quiescence by flow cytometry analyses and 5-ethynyl-20-deoxyuridine incorporation assays under normal and serum-starved conditions. RESULTS: NPSC quiescence was induced by 48 h of serum starvation, and they maintained quiescence for up to 21 days. Upon reactivation with serum, the quiescent NPSCs re-entered the cell cycle and exhibited enhanced clonogenic self-renewal, osteogenic differentiation and chondrogenic differentiation potentials compared to control NPSCs under normal culture conditions. We also found that autophagy underlay serum starvation-induced NPSC quiescence. Further study demonstrated that autophagy mediated the quiescence of NPSCs by regulating P27. CONCLUSIONS: Serum starvation efficiently induces quiescence in NPSCs. Quiescent NPSCs maintain stem cell properties. Our study reveals that autophagy plays a role in maintaining NPSC quiescence and that autophagy mediates the quiescence of NPSCs by regulating P27. We conclude that the induction of quiescence in cultured NPSCs provides a useful model for the analysis of mechanisms that might be relevant to the biology of NPSCs in vivo. BioMed Central 2019-04-15 /pmc/articles/PMC6466800/ /pubmed/30987681 http://dx.doi.org/10.1186/s13287-019-1219-8 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. |
spellingShingle | Research Li, Bin Sun, Chao Sun, Jing Yang, Ming-hui Zuo, Rui Liu, Chang Lan, Wei-ren Liu, Ming-han Huang, Bo Zhou, Yue Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27 |
title | Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27 |
title_full | Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27 |
title_fullStr | Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27 |
title_full_unstemmed | Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27 |
title_short | Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27 |
title_sort | autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of p27 |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466800/ https://www.ncbi.nlm.nih.gov/pubmed/30987681 http://dx.doi.org/10.1186/s13287-019-1219-8 |
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