An improved, standardised protocol for the isolation, enrichment and targeted neural differentiation of Nestin+ progenitors from adult human dermis.
Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.
Ernst
Nancy
N
Department of Dermatology, Allergology and Venerology, University of Lübeck, Lübeck, Germany.
Tiede
Stephan
S
Tronnier
Volker
V
Kruse
Charli
C
Zechel
Christina
C
Paus
Ralf
R
D000199:Actins / Q000378:metabolism; D000328:Adult; D053687:Adult Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D000368:Aged; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D003470:Culture Media / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D020405:Dermis / Q000166:cytology*; D005260:Female; D005353:Fibronectins / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D008297:Male; D008648:Mesoderm / Q000166:cytology; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D064231:Nestin; D016900:Neurofilament Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism
D000199:Actins; D003470:Culture Media; D016895:Culture Media, Serum-Free; D005353:Fibronectins; D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D016900:Neurofilament Proteins