Large-Scale Expansion and Characterization of Human Adult Neural Crest-Derived Multipotent Stem Cells From Hair Follicle for Regenerative Medicine Applications

Abstract

Aim: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. Materials and Methods: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. Results: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2+Sox10+Nestin+CD73+CD90+CD105+CD140a+CD 140b+CD146+CD166+CD271+CD349+ CD34-CD45-CD56-HLA-DR-, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. Conclusion: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40–200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice.

Comparative Analysis of Biological Properties of Large-Scale Expanded Adult Neural Crest-Derived Stem Cells Isolated from Human Hair Follicle and Skin Dermis

“…The cultures of HF NCSCs were obtained according to explant method by Sieber-Blum et al [8] in our modification for human samples [45]. Briefly, two skin specimens per donor were excised from the scalp zone under local anesthesia by dermal punch knife (Dermo-Punch ø 4 mm, Sterilab, Italy).…”

“…Our protocol of directed multilineage differentiation assay for human adult NCSCs was described earlier in details [45]. Briefly, we used the following differentiation media and adhesive substrates: adipogenic differentiation medium: DMEM high glucose (4.5 g/l) (BioWest, France) supplemented with 10% FBS, 1 μ M dexamethasone, 50 μ M indomethacine, 250 μ M isobutylmethylxanthine, 5 μ g/ml insulin (all: Sigma-Aldrich, USA), and 5% horse serum (BioWest, France); osteogenic differentiation medium: DMEM low glucose (1.0 g/l) (BioWest, France) supplemented with 10% FBS, 100 nM dexamethasone, 10 mM β -glycerophosphate, and 50 μ g/ml ascorbate-2-phosphate (all: Sigma-Aldrich, USA); neuronal differentiation medium: neurobasal medium (Gibco, UK), 2% B27 supplement (Gibco, UK), 1% N2 supplement (Gibco, UK), 5 μ M ec32 synthetic retinoid (AMSBIO, UK), 1 μ M forskolin (Sigma-Aldrich, USA), 20 ng/ml NGF (PeproTech, USA), 20 ng/ml BDNF (PeproTech, USA), 20 ng/ml GDNF (PeproTech, USA), and 20 ng/ml IGF (Gibco, UK); glial differentiation medium: neurobasal (Gibco, UK) and DMEM:F12 (Gibco, UK) media in a ratio 1 : 1, 2% B27 supplement (Gibco, UK), 1% N2 supplement (Gibco, UK), 1 μ M ec32 synthetic retinoid (AMSBIO, UK), 5 μ M forskolin (Sigma-Aldrich, USA), 20 ng/ml Neuregulin-1 (Gibco, UK), 10 ng/ml PDGF-BB (PeproTech, USA), and 20 ng/ml IGF (Gibco, UK).…”

“…Therefore, selective isolation of adult NCSCs and their effective large-scale expansion are important technical issues for the successful applications for regenerative medicine purposes. Various techniques were applied for the selective isolation of adult NCSCs: fluorescence-activated cell sorting [6, 42], selective culturing conditions for growth as neurosphere-like structures [42, 43], explant technique [44, 45], etc.…”

“…Promising sources for the isolation of adult NCSCs are the SD and HF due to the come-at-able and minimally invasive biopsy procedure. Explant technique is traditionally used for isolation and large-scale expansion of adult NCSCs from human hair follicles [44, 45]. However, the main and significant drawbacks of explant method are high frequency of explant detachment, spontaneous outgrowth of keratinocytes from HF explants, and long-term primary culturing step to obtain sufficient therapeutic cell number for subsequent expansion step [44, 45].…”

“…Explant technique is traditionally used for isolation and large-scale expansion of adult NCSCs from human hair follicles [44, 45]. However, the main and significant drawbacks of explant method are high frequency of explant detachment, spontaneous outgrowth of keratinocytes from HF explants, and long-term primary culturing step to obtain sufficient therapeutic cell number for subsequent expansion step [44, 45]. The originally described and widely used method for isolation and expansion of SD NCSCs is based on their ability to form neurosphere-like structures in a specific serum-free growth medium and ultra-low attachment culture dishes [42, 43].…”