The Development Of Improved Methods For Skin-derived Precursors Culture

Skin-derived precursor cells(SKPs)are self-renewal and multipotent adult stem cells,which have crucial therapeutic potential in alopecia and wound healing.However,production of SKPs progeny with both quantities and sternness that satisfy clinical demands is the prerequisite for the therapeutic potential of SKPs.A promising approach is the culturing SKPs in stirred suspension bioreactors(SSBs).In the present study,the proliferation,morphology,sternness and de novo hair follicle regeneration ability of SKPs were comparatively analyzed between the culture in static and SSB over consecutive five passages.We demonstrated that SSB yield 1785-fold expansion of undifferentiated pluripotent SKPs over five passages.In SSB,the cell aggregates were markedly smaller,and the cell aging was delayed.Besides,SSB did maintain the characteristic of SKPs.Intriguingly,the biopotency of our SSB-expanded SKPs in vivo was evaluated by the hair follicle regeneration assay,the in vivo hair follicle regeneration efficiency of SSB-expanded SKPs was similar to static conditions.Furthermore,sternness maintenance of human SKPs in SSB was verified by using both donors' foreskins and scalp.Collectively,the data indicated that SSB could generate large numbers of SKPs while maintaining their inherent regenerative function,which represents a significant potential in the clinical application of SKPs-based cell therapy.SKPs are multipotent stem cells derived from the dermis.SKPs are able to induce hair follicle regeneration.It is considered to be seed cells for the thereby of the skin injury.Their application is limited by the instability of their property during culture expansion.In this study,we employed ?-Catenin overexpressed epidermal cell conditioned medium to treat SKPs and found that the proliferation rate of SKPs was increased.In addition,the expression levels of SKPs signature genes Akp2,Sox2,Bmp4,Nestinl,PDGFa,Clstn2,Trpsl and Versican were up-regulated.In addition,the activity of alkaline phosphatase(AP)was enhanced significantly which reflect the hair follicle inducing ability of SKPs.Our study indicates that factors released by epidermal cells facilitate the stability of SKPs properties.In this study,the important components in the conditioned medium were selected.Endothelin-1(ET-1)plays an important role in the proliferation and sternness maintain of SKPs.SKPs treated with endothelin-1 can provide seed cells with faster proliferation and stronger hair follicle regeneration ability during stem cell treatment.