Influence of cryostorage on stem cell population in human keratinocytes

Abstract

Background & Aim During many years living skin equivalent (LSE) development has been an actual topic in regenerative medicine. Immature phenotype and high clonogenic potential of keratinocytes is related to the ability of the cells to reconstruct a pluristratified epidermis demonstrating an optimal balance between proliferation and differentiation. Cryostorage becomes a crucial stage of LSE development which potentially can influence keratinocyte stem cell (KSC) population. Towards this end, we wanted to evaluate cell characteristics before and after cryostorage. Methods, Results & Conclusion RNA expression of hemidesmosomal genes (integrins α6, β4 and β1), specific for epidermal basal layer, was examined by rtPCR analysis of keratinocyte primary culture before (p0) and after cryostorage (p1 – 1 and 7 day). Cell percentage variation with KSC phenotype (CD71-/integrin β4+) before (p0) and after cryostorage (1day) was measured by flow cytometry. Protein expression of integrin α6 before (p0) and after cryostorage (1 and 3 day) was measured by Western blot. Keratinocyte clonogenic potential was assessed by Colony Forming Efficiency test (CFE%) using standard protocol (J.Rheinwald, H. Green) before cryostorage (p2) and after thawing (p1-first passage after thawing and p2- second passage after 3 or 10 days of p1 cultivation). At the first day after thawing 12.5% of all live cells attached to plastic. Among them there was a decrease in RNA expression of α6 and β1 integrins; while increase in RNA expression of α6 integrin was observed on day 7. Wherein protein content of integrin α6 was the same before and after (1 day) thawing, but decreased to day 3. Besides, we found a rising rate of cell percentage with KSC phenotype after thawing. In addition, we observed an increase of keratinocyte clonogenic potential after thawing (CFE%): holoclones (d〉400μm) number in p2 after 10 days post-thawing keratinocyte cultivation was significantly higher compared to that before cryostorage. In contrast, if p1 was cultured for 3 days after thawing only, keratinocytes’ clonogenic potential in p2 was lower than prior to preservation. CFE test if performed immediately after thawing revealed absence of colonies. Presumably, hemidesmosomal genes expression transiently decreases in keratinocytes after cryostorage. We suggest that selection of progenitor keratinocytes with high density of integrin α6 molecules on their surface occurs. Funded by IDB RAS government program.