Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny

Abstract

This protocol describes methods of isolating skin-derived precursors (SKPs) from rodent and human skin, and for generating and enriching Schwann cells from rodent SKPs. SKPs are isolated as a population of non-adherent cells from the dermis that proliferate and self-renew as floating spheres in response to fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). Their differentiation into Schwann cells and subsequent enrichment of these differentiated progeny involves culturing SKPs as adherent cells in the absence of FGF2 and EGF, but in the presence of neuregulins, and then mechanically isolating the Schwann cell colonies using cloning cylinders. Methods for expanding and characterizing these Schwann cells are provided. Generation of primary SKPs takes approximately 2 weeks, while differentiation of Schwann cells requires an additional 4-6 weeks.

Age-Dependent Depletion of Human Skin-Derived Progenitor Cells

“…Our main goal in this work was to analyze a well-characterized human progenitor cell pool and correlate its size in relation to body aging. Several laboratories have now reported the existence of a multipotent dermal precursor cell population, the skin-derived precursor (SKP) cells (reviewed by Miller and coworkers [20,41]). Initially derived from rodents, now several groups reported isolation of SKPs from human scalp, foreskin, arm, beard and chin, in subjects ranging from fetal to old age [22,23,30,31,34,[36][37][38][39].…”

Stem cells and models of astrocytomas

“…For instance, studies on the neurons and astrocytes that are differentiated from skin precursor cells have demonstrated them as being neuron-like or astrocyte-like with the expression of some terminally differentiated markers, however they are in fact non-functional. 30,31 .…”

Sensory recovery after cell therapy in peripheral nerve repair: effects of naïve and skin precursor-derived Schwann cells

“…35 Skin-derived precursor (SKP) cells were obtained from dermis of postnatal Day 2 Lewis rats and cultured according to published protocols. 6,46,47 Briefly, skin from the dorsal torso was minced in HBSS (Gibco) on ice, incubated for 45 minutes in 0.1% collagenase XI at 37°C, dissociated mechanically, washed in cold DMEM, and passed through a 40-mm cell strainer. Filtrate was centrifuged at 1200 rpm, and the pellet was triturated and resuspended in culture medium (DMEM:F12 3:1, 100 U/ ml penicillin and 100 mg/ml streptomycin, 0.25 mg/ml Fungizon, 1× B27 supplement, 20 ng/ml EGF, and 40 ng/ ml bFGF [all from Gibco]).…”

“…To induce differentiation toward Schwann cells, aggregates were triturated and replated on poly-d-lysine/laminincoated culture dishes (Corning) in SC medium (DMEM/ F12 supplemented with 100 U/ml penicillin and 100 mg/ ml streptomycin, 0.25 mg/ml Fungizon, 4 mM forskolin, 10 ng/ml heregulin-1b, and 1% N2 supplement [Gibco]), as described. 6,40 Media were changed every 3-4 days. …”