Sphere-Forming Capacity as an Enrichment Strategy for Epithelial-Like Stem Cells from Equine Skin
Cellular Physiology and Biochemistry
This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Abstract 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
... oing 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 (IHC) 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. Statistical analysis The statistical analysis for the sphere formation assay and the population doubling time was based on the mixed model with horse as random effect and medium, time, sphere cycle and the two-way interactions as fixed effects. For testing the difference in sphere forming efficiency between a pure and impure EpSC isolate, a fixed effects model was fitted with the number of seeded cells (10, 100 and 1000), isolate (pure vs impure) and their interaction as fixed effects factors. SAS Version 9.3 was used for the analyses (SAS/STAT Software, Version 9.3, SAS Institute Inc.). The relationship between the number of cell units/field and the number of spheres/field was determined by the Pearson correlation coefficient. Values are given as mean ± standard error and a global significance level of 5% was used. Population doubling time of EpSCs The population doubling time (PDT) of isolated skin-derived epithelial cells from fresh adherent cultures (i.e. not cryopreserved) was used as an indicator of self-renewal and proliferation potential and was calculated from P 1 up to P 10 ( Table 2 ). Our results indicate that there was no statistically significant (P = 0.319) difference in the population doubling time of the different passages. The PDT in the adherent cultures for all the passages was positive and varied between 0.89 + 0.13 days and 1.39 + 0.35 days (means + SD, n = 6; Table 2 ).