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Osmolarity controls the differentiation of adipose-derived stem cells into nucleus pulposus cells via histone demethylase KDM4B

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Abstract

Adipose-derived stem cells (ADSCs) are an ideal source of cells for intervertebral disc (IVD) regeneration, but the effect of an increased osmotic microenvironment on ADSC differentiation remains unclear. Here, we aimed to elucidate whether hyperosmolarity facilitates ADSC nucleus pulposus (NP)-like differentiation and whether histone demethylase KDM4B is involved in this process. ADSCs were cultured under standard and increased osmolarity conditions for 1–3 weeks, followed by analysis for proliferation and viability. Differentiation was then quantified by gene and protein analysis. Finally, KDM4B knockdown ADSCs were generated using lentiviral vectors. The results showed that increasing the osmolarity of the differentiation medium to 400 mOsm significantly increased NP-like gene expression and the synthesis of extracellular matrix (ECM) components during ADSC differentiation; however, further increasing the osmolarity to 500 mOsm suppressed the NP-like differentiation of ADSCs. KDM4B, as well as the IVD formation regulators forkhead box (Fox)a1/2 and sonic hedgehog (Shh), were found to be significantly upregulated at 400 mOsm. KDM4B knockdown reduced Foxa1/2, Shh, and NP-associated markers’ expression, as well as the synthesis of ECM components. The reduction in NP-like differentiation caused by KDM4B knockdown was partially rescued by Purmorphamine, a specific agonist of Shh. Moreover, we found that KDM4B can directly bind to the promoter region of Foxa1/2 and decrease the content of H3K9me3/2. In conclusion, our results indicate that a potential optimal osmolarity window might exist for successful ADSC differentiation. KDM4B plays an essential role in regulating the osmolarity-induced NP-like differentiation of ADSCs by interacting with Foxa1/2-Shh signaling.

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Abbreviations

ADSC:

Adipose-derived stem cell

CCK-8:

Cell counting kit-8

DMEM:

Dulbecco’s modified Eagle’s medium

ECM:

Extracellular matrix

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

Fox:

Forkhead box

GPC3:

Glypican 3

IVD:

Intervertebral disc

KRT19:

Keratin 19

LBP:

Lower back pain

MSC:

Mesenchymal stem cell

NP:

Nucleus pulposus

PI:

Propidium iodide

RT-qPCR:

Reverse transcriptase quantitative polymerase chain reaction

SD:

Standard deviation

Shh:

Sonic hedgehog

TGF-β:

Transforming growth factor-β

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Funding

This study was partly supported by grants from the National Nature Science Foundation of China (81902279), the Nature Science Foundation of Zhejiang Province (LQ19H060002 and LQ19H160041) and the Medical and Health Science and Technology Project of Zhejiang Province (2018KY089 and 2020KY143).

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  1. Yujie Zhang and Yanyan Wang have contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, China

    Yujie Zhang, Xiaopeng Zhou, Jingkai Wang, Mingmin Shi, Jian Wang, Fangcai Li & Qixin Chen

  2. Department of Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, Zhejiang, China

    Yanyan Wang

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  1. Yujie Zhang
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Contributions

YJZ, YYW, QXC and FCL conceived and designed the research; YJZ, YYW, XPZ, JKW and MMS performed the research; YJZ, YYW and JW analyzed the data; QXC and FCL contributed with reagents or analytic tools; YJZ and YYW wrote the manuscript; and all authors revised and approved the manuscript.

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Correspondence to Fangcai Li or Qixin Chen.

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Zhang, Y., Wang, Y., Zhou, X. et al. Osmolarity controls the differentiation of adipose-derived stem cells into nucleus pulposus cells via histone demethylase KDM4B. Mol Cell Biochem 472, 157–171 (2020). https://doi.org/10.1007/s11010-020-03794-8

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