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

Abstract

Introduction. The adult neural crest-derived stem cells (NCSCs) have significant perspectives for use in regenerative medicine. The most attractive sources for adult NCSC isolation are the hair follicles (HF) and skin dermis (SD) because of easy access and minimally invasive biopsy. The aim of this study was to compare the biological properties of HF- and SD-derived NCSCs after their large-scale expansion. Methods. The conventional explant method was used to obtain HF NCSCs. For the isolation of SD NCSCs, a new combined technique consisting of preplating and subsequent culturing in 3D blood plasma-derived fibrin hydrogel was applied. The studied cells were characterized by flow cytometry, ICC, qPCR, Bio-Plex multiplex assay, and directed multilineage differentiation assays. Results. We have obtained both adult SD and HF NCSCs from each skin sample (n=5). Adult SD and HF NCSCs were positive for key neural crest markers: SOX10, P75 (CD271), NESTIN, SOX2, and CD349. SD NCSCs showed a higher growth rate during the large-scale expansion compared to HF NCSCs (p<0.01). Final population of SD NCSCs also contained more clonogenic cells (p<0.01) and SOX10+, CD271+, CD105+, CD140a+, CD146+, CD349+ cells (p<0.01). Both HF and SD NCSCs had similar gene expression profiling and produced growth factors, but some quantitative differences were detected. Adult HF and SD NCSCs were able to undergo directed differentiation into neurons, Schwann cells, adipocytes, and osteoblasts. Conclusion. The HF and SD are suitable sources for large-scale manufacturing of adult NCSCs with similar biological properties. We demonstrated that the NCSC population from SD was homogenous and displayed significantly higher growth rate than HF NCSCs. Moreover, SD NCSC isolation is cheaper, easier, and minimally time-consuming method.

Effect of the deuterium on efficiency and type of adipogenic differentiation of human adipose-derived stem cells in vitro

“…presented in previous studies 46 . Briefly, to confirm the osteogenic and adipogenic differentiation, the cells were fixed for 20 min in 10% buffered formalin (Sigma, USA), washed with DPBS (Sigma-Aldrich, USA) and stained for 20 min with 2% solution of Alizarin Red S (pH 4.1; for detecting calcified extracellular matrix deposits) or 0.5% solution of Oil Red O (for staining of neutral lipids) and Romanowsky-Giemsa stain for counterstaining, respectively (all -Sigma-Aldrich, USA).…”

“…The ADSCs were isolated from the lipoaspirate by enzymatic digestion in 0.1% collagenase IA and 0.1% pronase with 2% fetal bovine serum (FBS) (all -Sigma-Aldrich, USA) for 1 h at 37 °С. Detailed description of the method was presented in previous studies [40][41][42][43][44][45][46] . The obtained cell suspension was transferred to 25 cm 2 cell culture flask (SPL, Korea) and cultured in the following growth medium: modified МЕМ-α (Sigma-Aldrich, USA) prepared from the powder diluted with milliQ water of natural isotope content supplemented with 10% FBS (Sigma-Aldrich, USA), 2mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 ng/ml bFGF-2 (all from Sigma-Aldrich, USA).…”

“…BD FACS Diva 6.1 software (BD Pharmingen, BD Horizon USA) was used for analysis. Detailed description of the method was presented in previous studies 41,46 .…”

“…After 21 days fixation and staining of cells with Alizarin Red S were carried out on mineral deposits. Detailed description of the method was presented in previous studies 46 .…”

“…Further, the cell precipitate was cultured in a chondrogenic induction medium containing DMEM with 4.5 g/L glucose (BioWest, France) prepared from the powder by dissolution with milliQ water of natural isotope content supplemented with addition of 50 μg/ml ascorbate-2-phosphate, 40 μg/L L-proline, 100 μg/ml pyruvate sodium, 10 ng/ml rhTGF-β3, 10-7 M dexamethasone (all -Sigma-Aldrich, USA), 1% ITS supplement (all -Gibco, USA), 1.25 mg/ml of bovine serum albumin, BSA (BioWest, France). Detailed description of the method was presented in previous studies 46 .…”