Sphere-Forming Capacity as an Enrichment Strategy for Epithelial-Like Stem Cells from Equine Skin
Sphere-Forming Capacity as an Enrichment Strategy for Epithelial-Like Stem Cells from Equine Skin
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DOI：
10.1159/000366338
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发表时间：
2014-01-01
影响因子：
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通讯作者：
Spaas, Jan H.
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文献类型：
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作者：
Borena, Bizunesh M.;Meyer, Evelyne;Spaas, Jan H.
背景：哺乳动物皮肤在许多生命维持过程中起着关键作用，因此广泛的损伤可能危及生命。生理性皮肤再生是通过表皮和毛囊内持续的体细胞干细胞分化来实现的。然而，在严重的病理情况下，如烧伤创面、慢性创面和溃疡，内源性修复机制可能不足。因此，外源性纯化和扩增上皮样干细胞(EPSCs)可能有助于这些皮肤病的治疗。然而，关于利用悬浮培养分离、纯化和鉴定EPSCs的报道很少。方法：采集6匹母马的皮肤，分离纯化EPSCs。除了基于表型和功能特性的特征外，在分离时，即在第0代(P-0)、早期(P-4)和晚期(P-10)，使用不同的培养条件来评估球体的形成。结果：平均0.53+/-0.28%的原代皮肤来源细胞具有形成球体的能力，因此具有干细胞特性。此外，在EPSC培养液中观察到的球体明显多于分化培养液中的球体，证实了EPSC在悬液中存活的特权能力。此外，随着时间的推移以及随后的传代，每个球体的细胞数量显著增加。免疫表型分析发现，推测的EPSCs共表达细胞角蛋白(CK)14、酪蛋白激酶2β和主要组织相容性复合体(MHC)I，不表达泛型CK和广泛型CK。仅有少数细胞表达MHC II。根据CK 14、酪蛋白激酶2β、PAN CK和Wide CK的共同表达，证实了它们向角质形成细胞(P-4和P-10)的分化。在六个分离株中的一个，在贴壁培养中发现了非EPSC细胞类型。虽然形态特征和免疫组织化学(HC)证实为角质形成细胞表型，但以超低克隆密度(1和10个/cm(2))接种细胞可获得纯化，但球体形成率明显低于纯EPSCs(P=0.0012)。结论：本研究证明球体形成是分离纯化EPSCs的一种有价值的工具，并评估了在不同克隆接种密度下对清除细胞污染的效果。版权所有(C)2014年S.Karger AG，巴塞尔
Background: Mammal skin plays a pivotal role in several life preserving processes and extensive damage may therefore be life threatening. Physiological skin regeneration is achieved through ongoing somatic stem cell differentiation within the epidermis and the hair follicle. However, in severe pathological cases, such as burn wounds, chronic wounds, and ulcers, the endogenous repair mechanisms might be insufficient. For this reason, exogenous purification and multiplication of epithelial-like stem/progenitor cells (EpSCs) might be useful in the treatment of these skin diseases. However, only few reports are available on the isolation, purification and characterization of EpSCs using suspension cultures. Methods: In the present study, skin was harvested from 6 mares and EpSCs were isolated and purified. In addition to their characterization based on phenotypic and functional properties, sphere formation was assessed upon isolation, i.e. at passage 0 (P-0), and at early (P-4) and late (P-10) passages using different culture conditions. Results: On average 0.53 +/- 0.28% of these primary skin-derived cells showed the capacity to form spheres and hence possessed stem cell properties. Moreover, significantly more spheres were observed in EpSC medium versus differentiation medium, corroborating the EpSCs' privileged ability to survive in suspension. Furthermore, the number of cells per sphere significantly increased over time as well as with subsequent passaging. Upon immunophenotyping, the presumed EpSCs were found to co-express cytokeratin (CK) 14, Casein kinase 2 beta and Major Histocompatibility Complex (MHC) I and expressed no pan CK and wide CK. Only a few cells expressed MHC II. Their differentiation towards keratinocytes (at P-4 and P-10) was confirmed based on co-expression of CK 14, Casein kinase 2 beta, pan CK and wide CK. In one of six isolates, a non-EpSC cell type was noticed in adherent culture. Although morphological features and immunohistochemistry (HC) confirmed a keratinocyte phenotype, this culture could be purified by seeding the cells in suspension at ultralow clonal densities (1 and 10 cells/cm(2)), yet with a significantly lower sphere forming efficiency in comparison to pure EpSCs (P = 0.0012). Conclusion: The present study demonstrated sphere formation as a valuable tool to purify EpSCs upon their isolation and assessed its effectiveness at different clonal seeding densities for eliminating a cellular contamination. Copyright (C) 2014 S. Karger AG, Basel