Isolation, Characterization, and Safety Evaluation of Human Skin-Derived Precursors from an Adherent Monolayer Culture System

Abstract

Background. Skin-derived precursors (SKPs) are promising candidates for regenerative medicine. Several studies have transcultured human SKPs (termed tSKPs) from fibroblasts (FBs) expanded in monolayer culture. Herein, we optimized the procedure by treating flasks with poly-2-hydroxyethyl methacrylate (poly-HEMA). Methods. tSKPs generated from our adherent monolayer culture system were investigated for protein expression and differentiation capacity. The aggregated cells and the proliferative cells within tSKP spheres were detected by mix-culturing FBs expressing two different fluorescent proteins and BrdU- or EdU-positive cells, respectively. To distinguish tSKPs from FBs, we compared their phenotypes and transcriptomes. The tumorigenicity of tSKPs and FBs was also assessed in our study. Results. tSKPs expressed Versican, Fibronectin, Vimentin, Sox2, and Nestin. Under appropriate stimuli, tSKPs could differentiate to mesenchymal or neural lineages. While these spheres were heterogeneous populations consisting of both proliferative and aggregated cells, the rate of proliferative cells correlated with a seeding density. tSKPs, isolated from FBs, were distinctive from FBs in cell cycle, marker expression, neural differentiation potential, and transcript profiles despite the two sharing partial similarity in certain properties. As for tumorigenesis, both tSKPs and FBs could be considered as nontumorigenic ex vivo and in vivo. Conclusion. tSKPs were heterogeneous populations presenting similar characteristics as traditional SKPs, while being different from FBs. The potential mixture of FBs in spheres did not affect the biosafety of tSKPs, as both of which had normal karyotype and nontumorigenicity. Taken together, we suggested tSKPs had potential applications in regenerative medicine.

The minimal promoter (P1) of Xist is non-essential for X chromosome inactivation

“…Rabbit skin fibroblasts were isolated according to the previously described methods [28]. The skin was isolated from female WT and Xist P1 −/rabbits and cut into 0.5-mm 3 pieces.…”

Comparative transcriptome analysis of transcultured human skin-derived precursors (tSKPs) from adherent monolayer culture system and tSKPs-derived fibroblasts (tFBs) by RNA-Seq

“…The protocol for isolating tSKPs has been described previously in our study (6). For directing tSKPs into tFBs, spheres were collected, resuspended in dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum, and then seeded into cell culture dishes.…”

“…Conventionally, SKPs are cultured as floating spheres in suspending serum-free medium with required growth factors (3); however, many studies have reported that isolating human SKPs by this protocol has many limitations such as relatively low yield (4), slow growth rate (3), and heterogeneous spheres (5). Therefore, it is unsurprising that several studies have demonstrated a new technique to transculture human SKPs from adherent monolayer culture system (henceforth termed tSKPs), and these tSKPs are similar to traditional SKPs in morphology and function (6)(7)(8). Developments in tSKPs research have indicated their potential application in regenerative medicine.…”

“…Under proper stimulus, tSKPs can differentiate into cells of both neural and mesodermal lineages, including neurons (7), Schwann cells (6,7), smooth muscle cells (6,8), osteogenic (6,7) and adipogenic cells (6,7). It has also been reported that tSKPs can differentiate into fibroblasts (FBs) (8).…”

Cells, growth factors and biomaterials used in tissue engineering for hair follicles regeneration

“…1 ). Current techniques for cell isolation and culture in vitro make it possible to obtain potential cells, such as dermal papilla cells (DPCs), skin-derived precursors (SKPs), and hair follicle stem cells (HFSCs) [ [21] , [22] , [23] ]. These cells can be enmeshed in a biomaterial matrix, which mimics the chemical structure of HF extracellular matrix (ECM) and provides specific effects in regulating cell behaviors, such as cell survival, proliferation, and differentiation [ 24 , 25 ].…”