Neuronal and glial differentiation potential of skin-derived precursors is of great interest for clinical application in the treatment of neurodegenerative disease. In this sense, the pig model is a great candidate for the development of preclinical models. To the date, skin-derived precursor spheres have not been isolated from adult porcine skin. In order to optimize the protocol for isolating dermal precursor spheres from adult porcine skin, 15 porcine skin biopsies were subjected to three different processing protocols. Liberase-based digestion of ventral porcine skin gave rise to more cells with spherogenic capacity than other protocols and these spheres presented phenotypic and differentiation potential consistent with bona fide skin-derived precursor cells.
Skin-derived precursors (SKPs) are a population of neural crest-derived multipotent precursor cells present in both human and mouse dermis, distinct from mesenchymal and central nervous system stem cells [1-3]. They express genes characteristic of embryonic neural crest cells [
Neuronal and glial differentiation potential of SKPs is of great interest for clinical application in the treatment of neurodegenerative disease. However, development of safe clinical protocols usually requires preclinical validation of the in vitro results both in lower mammals (i.e rodents) and in a large animal model of disease. In this sense, the pig skin shares many physiological and anatomopathological similarities to its human counterpart. Dermal-epidermal thickness ratio ranges are very similar and both swine and human skin present well-developed rete ridges, dermal papillary bodies and abundant subcutaneous adipose tissue. Pigs and humans have relatively sparse body hair which progresses through the hair cycle independently of neighbouring follicles and, in contraposition to rodents, the mechanism of closure of partialthickness wounds proceeds largely through reepithelialization and not by wound contraction as in smaller mammals [
In 2004, Dyce et al. presented the first report of skinoriginated stem cells isolated from non-rodent animals [
A total of 15 skin biopsies (
weight. Biopsies were stored in PBS or RPMI (Sigma) supplemented with 2% penicillin/streptomycin (P/S) and processed at a maximum 24 hours post-sacrifice, as previously described [
For cell differentiation, 2000 cells were plated per well onto coverslips covered with extracellular matrix secreted by the 804G cell line [
Spheres were seeded in 12-mm diameter-coverslips previously treated to improve adherence as above. Two hours later, dermospheres were fixed with 4% paraformaldehyde for 15 minutes and permeabilised in 0.2% Triton X-100 and 6% FBS for 40 minutes. Differentiated cell cultures were fixed with 4% paraformaldehyde in PBS for 10 minutes and permeabilised in 0.5% Triton X- 100 for 10 minutes. Anti-nestin (Santa Cruz, polyclonal), vimentin (Sigma, clone v9), fibronectin (Sigma, polyclonal), SMA (Sigma, clone 1A4) and bIII tubulin (Abcam, polyclonal) primary antibodies were used. Goat anti rabbit Alexa 488 (H + L) and Goat anti mouse Alexa 488 (H + L) (Molecular Probes) were used as secondary antibodies. Hoechst 33258 (Sigma, 1 µg/ml) was used as nuclear counterstain and the slides were mounted on Vectashield (Vector laboratories).
To estimate percent cells differentiated to a given phenotype, 10 fields were counted per coverslip. Total cells were estimated through nuclear counts and cells in a given phenotype through counting of cells positive to the specific markers. Average of the 10 fields was calculated for each coverslip.
One-way ANOVA tests, with Scheffé corrections, were used to calculate p values between different protocols. IBM SPSS Statistics Version 20 was used.
In order to assess the best protocol for isolating dermal precursor spheres from adult porcine skin, we subjected 15 porcine skin biopsies (
and the disaggregating enzyme of choice (
On average, protocol 3 yielded increased cellularity (
However, it was more important to see if the obtained cells give rise to spheres, which are enriched in precursor cells. Protocol 1 yielded no sphere (0/3 biopsies). Protocol 2 generated spheres in 71.43% of the cases (5/7 biopsies). Cells obtained under protocol 3 gave rise to spheres in 100% of the cases (5/5 biopsies).
These results suggest that processing adult porcine ventral skin with Liberase DH generates SKP cultures in a reproducible manner, while ensuring maximum cellular yields are obtained per gram of tissue.
To characterize the porcine adult SKPs (pSKPs) obtained by using the protocol of choice (protocol 3), porcine dermospheres (
To verify their multipotent differentiation capacity, pSKPs were put into differentiation in basal medium (consisting of DMEM/F12 supplemented with B27, P/S, glutamine and 1% FBS) and in neural differentiation medium (consisting of DMEM/F12 supplemented with P/S, glutamine, N2, forskolin, and Heregulin β) for 14 days (see Matherials and Methods). Differentiated pSKPs expressed both mesodermal (SMA, 16.4%) and neural differentiation markers (bIII tubulin, 25.2%;
We have optimized the isolation of spherogenic cells from adult porcine skin. We conclude that liberase-based digestion of ventral porcine skin yields more cells with spherogenic capacity than other protocols and that these spheres presented phenotypic and differentiation potential consistent with bona fide skin-derived precursor cells.
This work was financed by grants provided by the Department of Industry, Innovation, Commerce and Tourism of the Basque Government (Gaitek 10/002), Diputación Foral de Gipuzkoa (OF 53/2011) and Ministerio de Ciencia e Innovación (PI10/02871 and INNPACTO programs, IPT-300000-2010-17). A.I. was supported by the “Programa I3SNS” (CES09/015) from Instituto de Salud Carlos III and by Osakidetza (Spain).