Mesenchymal Stem Cells Isolated from Equine Hair Follicles Using a Method of Air-Liquid Interface

Abstract

Equine mesenchymal stem cells (MSC) of various origins have been identified in horses, including MSCs from the bone marrow and adipose tissue. However, these stem cell sources are highly invasive in sampling, which thereby limits their clinical application in equine veterinary medicine. This study presents a novel method using an air-liquid interface to isolate stem cells from the hair follicle outer root sheath of the equine forehead skin. These stem cells cultured herewith showed high proliferation and asumed MSC phenotype by expressing MSC positive biomarkers (CD29, CD44 CD90) while not expressing negative markers (CD14, CD34 and CD45). They were capable of differentiating towards chondrogenic, osteogenic and adipogenic lineages, which was comparable with MSCs from adipose tissue. Due to their proliferative phenotype in vitro, MSC-like profile and differentiation capacities, we named them equine mesenchymal stem cells from the hair follicle outer root sheath (eMSCORS). eMSCORS present a promising alternative stem cell source for the equine veterinary medicine.

Keywords: Autologous veterinary therapy; Equine hair follicles; Mesenchymal stem cells; Minimal-invasive cell source; Tri-lineage differentiations.

Fig. 1

Histological structure of equine forehead skin and eMSCORS isolation using air-liquid interface method (n = 6). A Cross-section of forehead skin stained with H&E to study the anatomical structure of the equine donor skin. Sebaceous glands and the bulge region of the ORS were observed in the upper distal hair follicle in the dermis (B, yellow arrows). C, D A forehead skin (3 cm × 5 cm, large size for presentation) was dissected with its subcutaneous tissue removed, and sliced into long strips. E After collagenase treatment, the forehead skin dermis was loosened and hair follicles were plucked downwards from the side edge of the skin. F, G Hair follicles were transferred onto a Transwell membrane, and spindle-shaped cells migrated from the ORS onto the mesh, forming a cell layer; enlarged area of the ORS outgrowth, shown in G. Magnification: A a mosaic photo stitched from several photos in 4x by Keyence microscope, B, D 4x, E, F, G 10x

Fig. 2

In vitro cultivation and cell mobility properties of eMSCORS and eADMSC (n = 3). A Confluent cell layer of eADMSC after being isolated and cultivated in P1. B Confluent cell layers of eMSCORS exhibited an elongated dendritic-like morphology in P1. C eMSCORS formed aggregated colonies after being subcultured from the Transwell meshes into cell culture flasks in P0. D Cell yields per isolation of eADMSC and eMSCORS at P1. E, F Euclidean distance and movement velocity of eADMSC and eMSCORS. Statistical significance: * p < 0.05, *** p < 0.001

Fig. 3

Analysis of MSC-related biomarkers expressed in horse cells using flow cytometry (n = 3). To characterize the phenotypes of isolated eMSCORS and eADMSCS, cells were stained with fluorescently-labeled antibodies against the surface markers, according to the MSC marker definition panel. Cell populations of eMSCORS and eADMSCS were displayed and gated in the plot graph of forward scatter (FSC) versus side scatter (SSC). The expression of MSC-positive markers (CD29, CD44, CD90) and negative markers (CD14, CD34, CD45) are indicated by fluorescence intensity (blue) against the isotype control (red). Representative plot graphs and histograms are shown for eMSCORS (A) and eADMSC (B)

Fig. 4

Tri-lineage differentiation of eMSCORS and eADMSCS (n = 3). A-H Chondrogenic differentiation was achieved after 3 weeks of pellet culture; the cartilaginous pellets of Emscors (A,C,E,G) and eADMSCS (B,D,F,H) were stained with H&E (A, B), Alcian Blue (C, D), Safranin O (E, F), and underwent immune-detection of collagen type II (G, H). Enlarged photos of eMSCORS with higher magnifications are shown in A’, C’, E’, G’, and eADMSC in B’, D’, F’, H’. Osteogenic differentiation was induced for 3 weeks; cells were stained with BCIP/NBT to show ALP activity (I, K), and stained with Alizarin Red (J, L) to detect calcium deposition in eMSCORS (I,J) and eADMSCS (K, L). After adipogenic differentiation, morphological changes were observed in eMSCORS (M, white arrows) and eADMSCS (N). Magnifications: (A-M) 10x, (A’-H’) 40x, (N) 20x