Abstract

Skin burn repair requires efficient regeneration in order to achieve proper healing. We applied a sequential culturing system 2D1w/3D2w protocol for differentiating human mesenvhymal stem cell derived from adipose tissue (hMSC-AT) into epithelial-like stem cells (ELSC). hMSC-AT were cultured, characterized by immunophenotyping, mesenchymal differentiation and induced in a 2D culture using epithelial differentiation medium containing ATRA, ITS, dexamethasone, EGF and FGF-2 for 7 days (2D1w), followed by seeding in a 3D culture using chitosan hydrogel combined with the same cocktail (3D2w) for 2 weeks. The immunostaining of the cells was done at different stages of induction in order to characterize the differentiated cells using pan cytokeratin and anti-cytokeratins 14 and 18 antibodies. The expression of cytokeratins 5, 10, 14, 18 and 19 was evaluated by RT-PCR. The toxicity of chitosan hydrogel and proliferation of ELSC in 3D chitosan hydrogel were evaluated by MTT test. The viability of ELSC in 3D culture was evaluated by acridine orange/propidium iodide staining. The hMSC-AT were immunopositive to CD90, CD105, CD73, CD45 and CD34. The ELSC in the 2D culture expressed cytokeratin 19 after 7 days, while the other cytokeratins were not expressed. At the second week (3D culture), all of the above markers were expressed except cytokerstin10, moreover, the viability and proliferation were 98.02 ± 1.34 and 5.45 ± 0.36%, respectively. The cytotoxicity assay demonstrates the biocompatibility of chitosan hydrogel. The study reveals that hMSC-AT, seeded on chitosan hydrogel, can be induced into ELSC in the presence of other inducers.

中文翻译：

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用于从脂肪组织来源的人间充质干细胞生成上皮样干细胞的序贯培养系统

摘要

皮肤烧伤修复需要有效的再生才能实现适当的愈合。我们应用连续培养系统 2D1w/3D2w 方案将源自脂肪组织的人间膜干细胞 (hMSC-AT) 分化为上皮样干细胞 (ELSC)。培养 hMSC-AT，以免疫表型、间质分化为特征，并使用含有 ATRA、ITS、地塞米松、EGF 和 FGF-2 的上皮分化培养基在 2D 培养物中诱导 7 天 (2D1w)，然后使用壳聚糖接种到 3D 培养物中水凝胶与相同的混合物（3D2w）结合2周。在诱导的不同阶段对细胞进行免疫染色，以便使用全细胞角蛋白和抗细胞角蛋白 14 和 18 抗体来表征分化细胞。通过RT-PCR评估细胞角蛋白5、10、14、18和19的表达。通过MTT测试评价壳聚糖水凝胶的毒性和3D壳聚糖水凝胶中ELSC的增殖情况。通过吖啶橙/碘化丙啶染色评估 ELSC 在 3D 培养中的活力。hMSC-AT 对 CD90、CD105、CD73、CD45 和 CD34 呈免疫阳性。7天后，2D培养物中的ELSC表达细胞角蛋白19，而其他细胞角蛋白不表达。第二周（3D培养），除细胞角蛋白10外，上述所有标志物均表达，并且存活率和增殖率分别为98.02±1.34和5.45±0.36%。细胞毒性测定证明了壳聚糖水凝胶的生物相容性。研究表明，接种在壳聚糖水凝胶上的 hMSC-AT 在其他诱导剂存在的情况下可以被诱导为 ELSC。