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.

脂肪干细胞（ADSC）是椎间盘（IVD）再生的理想细胞来源，但是渗透微环境增加对ADSC分化的影响尚不清楚。在这里，我们旨在阐明高渗是否促进ADSC髓核（NP）样分化，以及组蛋白脱甲基酶KDM4B是否参与此过程。将ADSC在标准和增加的渗透压条件下培养1-3周，然后分析其增殖和生存力。然后通过基因和蛋白质分析对分化进行定量。最后，使用慢病毒载体产生了KDM4B组合式ADSC。结果表明，在ADSC分化过程中，将分化培养基的渗透压提高至400 mOsm会显着增加NP样基因的表达和细胞外基质（ECM）成分的合成。然而，进一步增加渗透压至500 mOsm抑制了ADSC的NP样分化。发现KDM4B以及IVD形成调节器叉头箱（Fox）a1 / 2和声波刺猬（Shh）在400 mOsm时显着上调。KDM4B组合式减少Foxa1 / 2，Shh和NP相关标记的表达，以及ECM成分的合成。由KDM4B敲除引起的NP样分化的减少可通过Shh的特异性激动剂Purmorphamine得以部分挽救。此外，我们发现KDM4B可以直接结合到Foxa1 / 2的启动子区域，并减少H3K9me3 / 2的含量。总之，我们的结果表明成功的ADSC分化可能存在潜在的最佳渗透压窗。KDM4B通过与Foxa1 / 2-Shh信号传导相互作用，在调节渗透性诱导的ADSC的NP样分化中起重要作用。