Li Shenyang, Ding Chengcheng, Guo Yonglong, Zhang Yanan, Wang Hao, Sun Xihao, Zhang Jun, Cui Zekai, Chen Jiansu
Aier School of Ophthalmology, Central South University, Changsha, China.
Aier Eye Institute, Changsha, China.
Front Bioeng Biotechnol. 2021 Sep 10;9:709488. doi: 10.3389/fbioe.2021.709488. eCollection 2021.
Suspended spheroid culture using ultralow attachment plates (ULAPs) is reported to effect corneal fibroblast reprogramming. Polydimethylsiloxane (PDMS), with hydrophobic and soft substrate properties, facilitates adherent spheroid formation that promotes cellular physical reprogramming into stem-like cells without using transcription factors. However, it is still unknown whether the biophysical properties of PDMS have the same effect on adult human corneal keratocyte reprogramming. Here, PDMS and essential 8 (E8) medium were utilized to culture keratocyte spheroids and fibroblast spheroids, and the reprogramming results were compared. We provide insights into the probable mechanisms of the PDMS effect on spheroids. qPCR analysis showed that the expression of some stem cell marker genes (, and ) was significantly greater in keratocyte spheroids than in fibroblast spheroids. The endogenous level of stemness transcription factors ( and ) was higher in keratocytes than in fibroblasts. Immunofluorescence staining revealed Klf4, Nanog, Sox2, ABCG2 and Pax6 were positively stained in adherent 3D spheroids but weakly or negatively stained in adherent 2D cells. Furthermore, , , , , , and gene expression was significantly higher in adherent 3D spheroids than in adherent 2D cells. Meanwhile, , and were more upregulated in adherent 3D spheroids than in suspended 3D spheroids. The RNA-seq analysis suggested that regulation of the actin cytoskeleton, TGFβ/BMP and HIF-1 signaling pathways induced changes in mechanotransduction, the mesenchymal-to-epithelial transition and hypoxia, which might be responsible for the effect of PDMS on facilitating reprogramming. In conclusion, compared to corneal fibroblasts, keratocytes were more susceptible to reprogramming due to higher levels of endogenous stemness transcription factors. Spheroid culture of keratocytes using PDMS had a positive impact on promoting the expression of some stem cell markers. PDMS, as a substrate to form spheroids, was better able to promote reprogramming than ULAPs. These results indicated that the physiological cells and culture conditions herein enhance reprogramming. Therefore, adherent spheroid culture of keratocytes using PDMS is a promising strategy to more safely promote reprogramming, suggesting its potential application for developing clinical implants in tissue engineering and regenerative medicine.
据报道，使用超低附着板（ULAPs）进行悬浮球体培养可实现角膜成纤维细胞重编程。聚二甲基硅氧烷（PDMS）具有疏水和柔软的底物特性，有助于形成贴壁球体，可在不使用转录因子的情况下促进细胞物理重编程为干细胞样细胞。然而，PDMS的生物物理特性对成年人类角膜基质细胞重编程是否具有相同作用仍不清楚。在此，利用PDMS和Essential 8（E8）培养基培养角膜基质细胞球体和成纤维细胞球体，并比较重编程结果。我们深入探讨了PDMS对球体作用的可能机制。qPCR分析表明，一些干细胞标记基因（、和）在角膜基质细胞球体中的表达显著高于成纤维细胞球体。角膜基质细胞中干性转录因子（和）的内源性水平高于成纤维细胞。免疫荧光染色显示，Klf4、Nanog、Sox2、ABCG2和Pax6在贴壁三维球体中呈阳性染色，但在贴壁二维细胞中呈弱阳性或阴性染色。此外，、、、、、和基因在贴壁三维球体中的表达显著高于贴壁二维细胞。同时，、和在贴壁三维球体中的上调程度高于悬浮三维球体。RNA测序分析表明，肌动蛋白细胞骨架、TGFβ/BMP和HIF-1信号通路的调节诱导了机械转导、间充质向上皮转化和缺氧的变化，这可能是PDMS促进重编程作用的原因。总之，与角膜成纤维细胞相比，角膜基质细胞由于内源性干性转录因子水平较高，更容易发生重编程。使用PDMS进行角膜基质细胞球体培养对促进一些干细胞标记物的表达具有积极影响。作为形成球体的底物，PDMS比ULAPs更能促进重编程。这些结果表明，本文中的生理细胞和培养条件可增强重编程。因此，使用PDMS进行角膜基质细胞贴壁球体培养是一种更安全地促进重编程的有前景的策略，表明其在组织工程和再生医学中开发临床植入物的潜在应用。
