Canine epidermal neural crest stem cells: characterization and potential as therapy candidate for a large animal model of spinal cord injury

Abstract

The discovery of multipotent neural crest-derived stem cells, named epidermal neural crest stem cells (EPI-NCSC), that persist postnatally in an easy-to-access location-the bulge of hair follicles-opens a spectrum of novel opportunities for patient-specific therapies. We present a detailed characterization of canine EPI-NCSC (cEPI-NCSC) from multiple dog breeds and protocols for their isolation and ex vivo expansion. Furthermore, we provide novel tools for research in canines, which currently are still scarce. In analogy to human and mouse EPI-NCSC, the neural crest origin of cEPI-NCSC is shown by their expression of the neural crest stem cell molecular signature and other neural crest-characteristic genes. Similar to human EPI-NCSC, cEPI-NCSC also expressed pluripotency genes. We demonstrated that cEPI-NCSC can generate all major neural crest derivatives. In vitro clonal analyses established multipotency and self-renewal ability of cEPI-NCSC, establishing cEPI-NCSC as multipotent somatic stem cells. A critical analysis of the literature on canine spinal cord injury (SCI) showed the need for novel treatments and suggested that cEPI-NCSC represent viable candidates for cell-based therapies in dog SCI, particularly for chondrodystrophic dogs. This notion is supported by the close ontological relationship between neural crest stem cells and spinal cord stem cells. Thus, cEPI-NCSC promise to offer not only a potential treatment for canines but also an attractive and realistic large animal model for human SCI. Taken together, we provide the groundwork for the development of a novel cell-based therapy for a condition with extremely poor prognosis and no available effective treatment.

Keywords: Adult stem cells; Canine epidermal neural crest stem cells; Dog model; EPI-NCSC; Hair follicle; Neural crest; Spinal cord injury; cEPI-NCSC.

Figure 1.

Anatomy of canine hair. (A): Dorsal view of canine haired skin. Skin is from an adult male castrated Dachshund. Representative image of canine haired skin. Note that each follicular unit has multiple hair shafts exiting through one follicular opening. (B): En face view of canine haired skin in (A) showing the anatomy of a compound hair (bracket). The primary hair (arrowhead) is prominent and surrounded by several smaller secondary hairs (e.g., arrow). (C): Dogs included in this study. Canine epidermal neural crest stem cells were isolated from all dogs.

Figure 2.

Bulge explant culture and ex vivo expansion. (A): Time course of ex vivo expansion of canine epidermal neural crest stem cells (cEPI-NCSC) from a primary hair follicle (dog 4: phase contrast optics). Scale bars = 400 μm. (B): cEPI-NCSC short-term ex vivo expansion, representative image. Cells start to divide less than 24 hours after plating. Millions of cells are obtained from one bulge explant by day 5 of ex vivo expansion (dog 3: phase contrast optics). Scale bars = 200 μm. (C): cEPI-NCSC growth curve during ex vivo expansion. On average, 3 million cells were obtained per hair follicle within 11 days after explantation.

Figure 3.

Comparative analysis of neural crest stem cell markers. (A): Neural crest marker gel electrophoresis of reverse transcriptase polymerase chain reaction results. Test of canine epidermal neural crest stem cell (cEPI-NCSC) molecular characterization and housekeeping primer sets in different dog tissues. (B): cEPI-NCSC gene expression levels relative to HKGs after ex vivo expansion. Percentage of relative expression by quantitative reverse transcriptase polymerase chain reaction of selected molecular signature genes (Msx2, Thop1, Ube4b, Ets1, Crmp1, Cryab, Adam12, Myo10), general neural crest stem cell genes (Snai2, Sox10), and a general stem cell gene (Nes). Data are from five donors with exception of Sox10 (n = 4). Abbreviations: -, water; -RT, minus reverse transcriptase control; B, brain; C, colon; E, embryo; EPI, canine epidermal neural crest stem cells; HKG, housekeeping gene; L, standard, 100–200 base pairs; O, ovary; P placenta; T, testicle; U, uterus.

Figure 4.

Pluripotency marker expression. (A): Test of canine epidermal neural crest stem cell (cEPI-NCSC) molecular characterization primer sets. (B): cEPI-NCSC gene expression after ex vivo expansion. Percentage of relative expression by quantitative reverse transcriptase polymerase chain reaction of pluripotency genes; data from four donors. (C): Characterization at the protein level by immunocytochemistry. cEPI-NCSC show immunofluorescence for the pluripotency markers Oct4 (images A, A′), Sox2 (images B, B′), Klf4 (images C, C′), c-Myc (images D, D′), Lin28 (images E, E′), and Nanog (images F, F′). Blue, DAPI nuclear stain. Data are from five donors. Scale bars = 50 μm. Abbreviations: -, water; -RT, minus reverse transcriptase; DAPI, 4′,6-diamidino-2-phenylindole; E, embryo; EPI, canine epidermal neural crest stem cells; HKG, housekeeping gene; L, standard, 100–200 base pairs.

Figure 5.

Characterization of canine epidermal neural crest stem cells (NCSC) by immunocytochemistry. Representative images. Canine epidermal NCSC express at the protein level NCSC signature genes, general neural crest markers, and a general stem cell marker. Images shown are the antibody-specific (black and white) and merged images (color) using DyLight 488- or DyLight 594-conjugated secondary antibodies. Blue, DAPI nuclear stain. (A, A′): Transcription factor Ets1 shows mainly nuclear immunoreactivity. (B, B′): Thop1 shows variable cytoplasmic immunoreactivity. (C, C′): Transcription factor Msx2 with mostly cytoplasmic immunoreactivity. (D, D′): Cytosolic protein Crmp1 has variable cytoplasmic expression. (E, E′): Ube4b shows strong cytoplasmic expression. (F, F′): Myo10 shows strong cytoplasmic immunoreactivity. (G, G′): Adam12, a membrane-anchored protein, shows variable cytoplasmic expression. (H, H′): Cryab has diffuse cytoplasmic immunoreactivity. (I, I′): The intermediate filament Nes shows cytoplasmic immunoreactivity. (J, J′): Sox10, a nucleo-cytoplasmic shuttle protein, shows mainly cytoplasmic immunoreactivity. Scale bars = 50 μm. Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6.

Clonal culture. Primary clone from haired skin of dog 1. (A): Clone-forming cell. Inset, clone-forming cell at higher magnification. (B): Six daughter cells of the same clone-forming cells at day 3. (C–E): Cells continued to proliferate, forming a clone consisting of thousands of cells by clonal culture day 9. (F): Growth curve of cells in clones showing exponential growth (n = 29). Abbreviation: cEPI-NCSC, canine epidermal neural crest stem cell.

Figure 7.

Indirect immunofluorescence of primary and secondary clones. Representative images. The clones were probed with cell-type-specific antibodies to determine whether primary and secondary clones contain more than one cell type. Triple stains combining two cell-type-specific antibodies (isolated red or green images) and merged image using DyLight 488- and DyLight 594-conjugated secondary antibodies and DAPI nuclear stain (blue). (A): Primary clones. Two cell types were observed within the clonal cultures. Immunoreactive COLII cell (image A′, arrow) that is not reactive for SMA (image A). Immunoreactive SMA cell (image B, arrowhead) that is not reactive for β-III tubulin (image B′). Immunoreactive βIII-tubulin cell (arrow) that is mostly not reactive for SMA (image B). Immunoreactive GFAP cell (image C, arrow) that is not reactive for SMA (image C′). Immunoreactive SMA cell (image C′, arrowhead) that is not reactive for GFAP (image C). GFAP-immunoreactive cell (image D, arrowhead) that is β-III tubulin immunonegative (image D′). Immunoreactive β-III tubulin (image D′, arrow) cell that is not reactive for GFAP (image D). The data show that canine epidermal neural crest stem cells are multipotent. Scale bars = 50 μm. (B): Secondary clones. Representative images. Immunocytochemistry combining two cell-type-specific antibodies using DyLight 488-conjugated (green fluorescence) and DyLight 594-conjugated (red fluorescence) secondary antibodies and DAPI nuclear stain (blue). Two cell types were observed within the secondary clonal cones. Immunoreactive COLII cell (image A, arrow) that is not reactive for SMA (image A′, arrow). Immunoreactive SMA cell (image B, arrowhead) that is not reactive for GFAP (image B′, arrowhead). Immunoreactive GFAP cell (image B′, arrow) that is not reactive for SMA (image B, arrow). Immunoreactive β-III tubulin cells (image C). These results show that canine epidermal neural crest stem cells can undergo self-renewal. Scale bars = 50 μm. Abbreviations: Col II, collagen type II; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; SMA, smooth muscle actin.