Small molecules facilitate single factor-mediated sweat gland cell reprogramming.
Small molecules facilitate single factor-mediated sweat gland cell reprogramming.
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小分子促进单因子介导的汗腺细胞重编程
DOI：
10.1186/s40779-022-00372-5
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发表时间：
2022-03-29
影响因子：
21.1
通讯作者：
Sun XY
中科院分区：
文献类型：
--
作者：
Ji SF;Zhou LX;Sun ZF;Xiang JB;Cui SY;Li Y;Chen HT;Liu YQ;Gao HH;Fu XB;Sun XY
大面积皮肤缺损会严重破坏整个皮肤结构，并会不可逆地损害汗腺（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，这可能提供一种有前途的方法，以获得理想的细胞类型，用于受损皮肤的功能修复和再生。采用定量PCR（qPCR）、免疫荧光和流式细胞术检测SG标志物细胞角蛋白5（CK 5）、细胞角蛋白10（CK 10）、细胞角蛋白18（CK 18）、癌胚抗原（CEA）、水通道蛋白5（AQP 5）和α-平滑肌肌动蛋白（α-SMA）的表达。进行钙活性分析以测试诱导的SG样细胞（iSGCs）的功能。还使用小鼠异种移植模型来评估iSGC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-碘汗液试验用于确认iSGCs的体内再生。EDA过表达驱动HDF在SG培养基（SGM）中转化为iSGCs。qPCR检测结果显示，iSGCs中SG标志物CK 5、CK 18和CEA的mRNA水平明显升高，流式细胞术检测结果显示，（4.18 ± 0.04）%的iSGCs表达CK 5，（4.36 ± 0.25）%的iSGCs表达CK 18。化学鸡尾酒的加入大大加速了SG命运计划。qPCR结果显示iSGCs中CK 5、CK 18和CEA的mRNA表达显著增加，以及导管标记物CK 10和管腔功能标记物AQP 5的活化。流式细胞仪检测结果显示，经化学混合物处理后，分别有（23.05 ± 2.49）%的iSGCs表达CK 5+，（55.79 ± 3.18）%的iSGCs表达CK 18+。钙活性分析显示，iSGCs对乙酰胆碱的反应性与原代SG细胞接近[（60.79 ± 7.71）% vs.（70.59 ± 0.34）%，ns]。体内移植实验显示约（5.2 ± 1.1）%的小鼠汗液试验阳性，组织学分析结果表明iSGCs处理的小鼠中存在再生SG结构。我们开发了一种SG重编程策略，通过使用单因子EDA与SGM和小分子的组合从HDF产生功能性iSGCs。iSGCs的产生对未来SG修复的原位皮肤再生具有重要意义。在线版本包含补充材料，可通过10.1186/s40779-022-00372-5获得。
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. The online version contains supplementary material available at 10.1186/s40779-022-00372-5.