背景：肌成纤维细胞（MYF）通常被认为是肺纤维化发病机制中细胞外基质过度沉积和疤痕形成的罪魁祸首。另一方面，脂成纤维细胞 (LIF) 是由其脂质储存能力定义的，主要存在于肺泡区域。它们被认为在肺纤维化中发挥保护作用。我们之前报道过在纤维化形成和消退过程中发生了 LIF 向 MYF 可逆分化转换。在本研究中，我们测试了 WI-38 细胞（一种人胚胎肺成纤维细胞系）是否可用于研究成纤维细胞向 LIF 或 MYF 表型的分化，以及这是否与特发性肺纤维化 (IPF) 相关。方法：使用 WI-38 细胞，使用 TGF-β1 处理触发成纤维细胞 (FIB) 向 MYF 分化，并使用二甲双胍处理触发 FIB 向 LIF 分化。我们还通过分别用 TGF-β1 或二甲双胍预处理 WI-38 细胞来分析 MYF 到 LIF 以及 LIF 到 MYF 的分化。我们使用 IF、qPCR 和bulk RNA-Seq 来分析细胞的表型和转录组变化。我们将来自 WI-38 细胞的体外转录组数据（通过批量 RNA 测序获得）与源自 IPF 细胞图谱的 LIF 和 MYF 的转录组特征以及我们自己的来自 IPF 患者肺的单细胞转录组数据相关联体外培养的成纤维细胞（LF-IPF）。我们还进行了肺泡球测定，以评估所提出的 LIF 和 MYF 细胞支持肺泡上皮 2 型细胞生长的能力。结果：WI-38 细胞和 LF-IPF 对 TGF-β1 和二甲双胍治疗表现出相似的表型和基因表达反应。对用 TGF-β1 或二甲双胍处理的 WI-38 细胞和 LF-IPF 进行批量 RNA-Seq 分析，结果显示相似的转录组变化。我们还展示了从 Habermann 等人提取的 LIF 和 MYF 签名的部分保守性。分别用二甲双胍或 TGF-β1 处理的 WI-38 细胞的 scRNA-seq 数据集。肺泡球测定表明，LIF 促进类器官生长，而 MYF 抑制类器官生长。最后，我们提供证据支持使用 WI-38 细胞进行 MYF 至 LIF 以及 LIF 至 MYF 可逆转换。结论：WI-38 细胞代表了一种通用且可靠的模型，可用于研究成纤维细胞分化为与肺纤维化形成和消退相关的 MYF 或 LIF 表型的复杂动态，为推动未来的研究提供有价值的见解。© 作者。

Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: Using WI-38 cells, Fibroblast (FIB) to MYF differentiation was triggered using TGF-β1 treatment and FIB to LIF differentiation using Metformin treatment. We also analyzed the MYF to LIF and LIF to MYF differentiation by pre-treating the WI-38 cells with TGF-β1 or Metformin respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IPF patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 cells and LF-IPF display similar phenotypical and gene expression responses to TGF-β1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 cells and LF-IPF treated with TGF-β1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-β1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the MYF to LIF and LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.© The author(s).