Research Center for Tissue Repair and Regeneration Affiliated To Medical Innovation Research Department and 4th Medical Center, PLA General Hospital and PLA Medical College; PLA Key Laboratory of Tissue Repair and Regenerative Medicine and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, 28 Fu Xing Road, Beijing, 100853, China.
Research Unit of Trauma Care, Tissue Repair and Regeneration, Chinese Academy of Medical Sciences, 2019RU051, Beijing, 100048, China.
Mil Med Res. 2022 Mar 29;9(1):13. doi: 10.1186/s40779-022-00372-5.
Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands (SG), thus impairing the skin's physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin.
The expression of the SG markers cytokeratin 5 (CK5), cytokeratin 10 (CK10), cytokeratin 18 (CK18), carcino-embryonic antigen (CEA), aquaporin 5 (AQP5) and α-smooth muscle actin (α-SMA) was assessed with quantitative PCR (qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells (iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs. BALB/c nude mice were randomly divided into a normal group, SGM treatment group and iSGC transplantation group. Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs.
EDA overexpression drove HDF conversion into iSGCs in SG culture medium (SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18 and CEA in iSGCs, and flow cytometry data demonstrated (4.18 ± 0.04)% of iSGCs were CK5 positive and (4.36 ± 0.25)% of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails, (23.05 ± 2.49)% of iSGCs expressed CK5 and (55.79 ± 3.18)% of iSGCs expressed CK18, respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79 ± 7.71)% vs. (70.59 ± 0.34)%, ns]. In vivo transplantation experiments showed approximately (5.2 ± 1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice.
We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.
大面积皮肤缺损严重破坏整体皮肤结构，并可不可逆地损伤汗腺（SG），从而损害皮肤的生理功能。本研究旨在开发一种逐步重编程策略，将成纤维细胞转化为 SG 谱系，这可能为获得理想的细胞类型以实现受损皮肤的功能修复和再生提供一种有前景的方法。
采用定量 PCR（qPCR）、免疫荧光和流式细胞术评估 SG 标志物细胞角蛋白 5（CK5）、细胞角蛋白 10（CK10）、细胞角蛋白 18（CK18）、癌胚抗原（CEA）、水通道蛋白 5（AQP5）和α-平滑肌肌动蛋白（α-SMA）的表达。钙活性分析用于测试诱导的 SG 样细胞（iSGCs）的功能。还使用小鼠异种移植模型来评估 iSGCs 的体内再生。将 BALB/c 裸鼠随机分为正常组、SGM 治疗组和 iSGC 移植组。免疫细胞化学分析和淀粉-碘汗试验用于确认 iSGCs 的体内再生。
EDA 过表达在 SG 培养基（SGM）中驱动 HDF 向 iSGCs 转化。qPCR 表明 iSGCs 中 SG 标志物 CK5、CK18 和 CEA 的 mRNA 水平显著增加，流式细胞术数据表明，iSGCs 中 CK5 阳性细胞的比例为（4.18±0.04）%，CK18 阳性细胞的比例为（4.36±0.25）%。化学鸡尾酒的添加大大加速了 SG 命运程序。qPCR 结果显示 iSGCs 中 CK5、CK18 和 CEA 的 mRNA 表达显著增加，以及导管标志物 CK10 和腔功能标志物 AQP5 的激活。流式细胞术表明，在用化学鸡尾酒处理后，iSGCs 中表达 CK5 的比例为（23.05±2.49）%，表达 CK18 的比例为（55.79±3.18）%。钙活性分析表明，iSGCs 对乙酰胆碱的反应性接近原代 SG 细胞[（60.79±7.71）% vs. （70.59±0.34）%，ns]。体内移植实验显示，约有（5.2±1.1）%的小鼠汗试验阳性，组织学分析结果表明，iSGC 治疗小鼠存在再生的 SG 结构。
我们开发了一种 SG 重编程策略，通过使用单因素 EDA 结合 SGM 和小分子从 HDF 中生成功能性 iSGCs。iSGCs 的产生对于未来基于 SG 恢复的原位皮肤再生具有重要意义。
