人表皮干细胞在创伤修复中的稳定性和生物安全性：临床前评价。
Stability and biosafety of human epidermal stem cell for wound repair: preclinical evaluation.
机构信息
Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China.
Department of Burn and Plastic Surgery, The First Affiliated Hospital of Wannan Medical College, Wuhu, 241001, Anhui, China.
出版信息
Stem Cell Res Ther. 2023 Jan 5;14(1):4. doi: 10.1186/s13287-022-03202-6.
BACKGROUND
Cell therapy is a key technology to prevent sacrificing normal skin. Although some studies have shown the promise of human epidermal stem cells (EpiSCs), the efficacy, biosafety and quality control of EpiSC therapy have not been systematically reported.
METHODS
The biosafety, stemness maintenance and wound repair of EpiSC were systematically verified by in vitro and in vivo experiments. EpiSC were prepared from the foreskin using a collagen type IV rapid adherence method. The EpiSCs were identified by flow cytometry, immunofluorescence staining and cell morphology. The well-growing passage 1 (P1) EpiSCs were used to determine the proliferation curve (counting method). EpiSC clone formation assay was performed by Giemsa staining. Nude mice were used to prepare a full-thickness skin defect wound model to detect the repair effect of EpiSCs. The biosafety of EpiSCs was double tested in vitro and in vivo.
RESULTS
The results showed that the expression of specific markers and clone formation efficiency was stable when passage 1 (P1) to P8 cells were cultured, and the stemness rate of P8 cells was close to 85.1%. EpiSCs were expanded in vitro for 25 days, the number of cells reached 2.5 × 10, and the transplantable area was approximately 75% of the total body surface area (TBSA). At 45 days, the total number of cells was approximately 30 billion, and the transplantable area was approximately the size of a volleyball court. A nude mouse wound model indicated that EpiSCs could rapidly close a wound. On postinjury day 7, the wound epithelialization rate in the cell transplantation group was significantly higher than that in the NaCl group (P < 0.05). In vitro, cell senescence increased, and telomerase activity decreased in P1 to P8 EpiSCs. In vivo, there were no solid tumors or metastatic tumors after EpiSC (P8) transplantation. In addition, the quality control of cultured cells met the clinical application criteria for cell therapy.
CONCLUSION
This preclinical study showed the stability and biosafety of human EpiSC therapy for wound repair.
背景
细胞疗法是预防牺牲正常皮肤的关键技术。虽然一些研究表明人类表皮干细胞（EpiSCs）具有前景，但 EpiSC 治疗的疗效、生物安全性和质量控制尚未得到系统报道。
方法
通过体外和体内实验系统验证 EpiSC 的生物安全性、干细胞维持和伤口修复。使用胶原 IV 快速黏附法从包皮中制备 EpiSC。通过流式细胞术、免疫荧光染色和细胞形态学鉴定 EpiSC。使用良好生长的第 1 代（P1）EpiSC 确定增殖曲线（计数法）。通过吉姆萨染色进行 EpiSC 克隆形成试验。使用裸鼠制备全层皮肤缺损创面模型，以检测 EpiSC 的修复效果。体外和体内双重复检 EpiSC 的生物安全性。
结果
结果表明，当培养到第 1 代（P1）至 P8 细胞时，特异性标志物的表达和克隆形成效率稳定，P8 细胞的干细胞率接近 85.1%。EpiSC 在体外培养 25 天，细胞数量达到 2.5×10，可移植面积约为体表面积（TBSA）的 75%。第 45 天，细胞总数约为 300 亿，可移植面积约为一个排球场大小。裸鼠创面模型表明 EpiSC 可迅速封闭创面。在细胞移植组，损伤后第 7 天，创面上皮化率明显高于 NaCl 组（P<0.05）。体外，P1 至 P8 EpiSC 中细胞衰老增加，端粒酶活性降低。体内，EpiSC（P8）移植后无实体瘤或转移瘤。此外，培养细胞的质量控制符合细胞治疗的临床应用标准。
结论
这项临床前研究表明，人类 EpiSC 治疗创面修复的稳定性和生物安全性。
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