Adipogenic Differentiation of Canine Hair Follicle Stem Cells (cHFSCs)

Stem cells are undifferentiated cells, defined by their ability at the single cell level to both self-renew and differentiate to produce mature progeny cells, including both non- renewing progenitors and terminally differentiated effector cells (Wagers and Weissman, 2004). The hair follicle was functioning as the ‘‘bone marrow of the skin’’ and continuously remodeled its cutaneous microenvironment including skin innervation and vasculature (Schmidt-Ullrich and Paus, 2005). Hair follicle stem cells (HFSC) were located at the bulge/isthmus region of the human hair follicles (Unna, 1876). The keratinocytes of the bulge region of mice hair follicles were quiescent and long-lived, multipotent (could give rise to all cell lineages of the hair follicle) and had high proliferative potential, thus fulfilling many of the traditional criteria for true stem cells (Lin and Andersen, 2008). Hair follicle stem cells were also characterized by expression of gene markers such as b1 integrin,cytokeratin 15, cytokeratin 19, a6 integrin, CD71, S100A, Caveolin1, CD200, PHLDA1, follistatin, frizzled homolog 1, nestin (neuron progenitor cell marker)  and CD34 glycoprotein in human (Ohyama et al., 2006).

The hair follicle bulge cells were thought to differentiate into hair matrix keratinocytes, medulla, cortex, cuticle and basal sebocytes of the sebaceous glands and epidermis which could be used for wound healing (Lavker et al., 1993). Murine HFSCs differentiated into epidermis and called them as bipotent cells.They can also differentiate into neurons, glial cells, keratinocytes and smooth muscle cells in vitro in mice (Amoh et al., 2004). The HFSCs were implanted into gap region of a severed sciatic nerve. The HFSCs greatly enhanced the rate of nerve regeneration and the restoration of nerve function. The follicle stem cells transdifferentiated largely into Schwann cells which were known to support neuron regrowth (Hoffman, 2006). Goat hair follicle stem cells (gHFSCs) were differentiated into osteocytes, chondrocytes and myocytes and differentiated osteocytes were confirmed for Von Kossa staining and expressed Osteocalcin. Differentiated chondrocytes were positive for Alcian blue staining and COL2A1 expression and myogenic induction confirmed by positive expression of gHFSCs for MyoG (He et al., 2016). Hence the present study was carried out to find the multipotent ability of canine hair follicle stem cells (cHFSCs) and differentiated as adipocytes.

Ethical approval
 
The research work has been carried out at the Department of Veterinary Anatomy, Madras Veterinary College, TANUVAS, Chennai during 2018-19 as per the approval of the Institutional ethical committee for stem cell research and therapy, Approval No.01/ICSCRT/2018 dated 23.11.2018 of the Chairman, IC-SCRT, Madras Veterinary College, Chennai-07.
 
Isolation and culture of cHFSCs
 
Canine skin samples were collected and washed with 2% chlorhexidine for 3 minutes and washed with 70% isopropyl alcohol for a minute (Hibbard et al., 2002). Canine hair follicles were separated from skin by digesting them in Dispase-II enzyme (neutral protease, grade II; Roche; Cat. No. 04942078001; 2.4 U/ml in DMEM) at 4^oC for overnight. In this method, to prepare single cell suspension, plucked canine hair follicles were placed into 0.25% trypsin-EDTA solution (SIGMA®; Cat. No. T4049) for digestion at 37^oC in CO₂ incubator for 15-20 minutes.Trypsin-EDTA was neutralized by adding serum and the digested bulge follicular cells were dissociated into single cell by gentle pipetting repeatedly and cultured in growth medium containing William’s E medium (Sigma- Aldrich; W0397) or DMEM supplemented with 10% fetal bovine serum(FBS) (Gibco®; Cat. No. 10082-147) and 10 ng/ml of epidermal growth factor (EGF) (Thermofisher;PHG0311), 5 ng/ml of insulin growth factor (IGF-I; Cat. No. I8779) two per cent ABAM (ABAM) (Gibco®; Cat.No.15240-062) and one per cent amphotericin B (Sigma- Aldrich;Cat. No. A2942) in a humidified atmosphere at 37^oC with 5 per cent CO₂. Culture plates were coated with collagen coating solution (CELL; Cat. No. 125-50) prior to seeding the cells. Cultures of cHFSCs were maintained upto passage 2 (P2) level for differentiation.

Cell proliferation(CD) and population doubling time (PDT) were calculated by following CD=In (Nf/Ni)/In 2(Nf- final cell number; Ni- initial cell number; In- natural logarithm) (Spencer et al., 2012); PDT= 1/r (r = log N₂- Log N₁/ t₂-t₁; N₂- total cells harvested at the hour of t₂. N₂ - total cells harvested at the hour of t₁) (Hilmi et al., 2013).
 
Immunocytochemistry
 
Cultured cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 5 minutes and incubated with 8% blocking buffer for 25 minutes. Fixed cells were incubated with 250 µl of primary antibody Anti-Cytokeratin 15 (CK15)(Cat. No. ab80522) (5 µg/ml) at 4^oC for overnight and washed further 250 µl of secondary antibody (Goat Anti-mouse IgG FITC conjugated) of 1:1000 dilutions was added and incubated for one hour and counterstained with DAPI. Washing with DPBS was followed after every step of incubation.
 
Viability assessment
 
Growth media was removed from cHFSCs culture plates and washed with DPBS. Cells were fixed with methanol for 5 minutes in room temperature. Fixative was removed and cells were washed with distilled water. Acridine orange stock solution was prepared by dissolving 1 g in 1000 ml of Dulbecco’s phosphate buffered saline (DPBS). For working solution, Acridine orange stock solution (5 ml) was mixed with 20ml of 0.1M Citric acid and 1.3 ml of 0.3 M Sodium heptahydrate. Working solution was poured over the cell colonies and incubated for five minutes in room temperature. Culture plates were washed and observed under 530 nm filter for green fluorescence and 600 nm for red fluorescence.

Adipogenic differentiation and staining
 
cHFSCs of passage 2 (P2) with 70% confluency were incubated with adipogenic differentiation medium (Stem Pro™ Adipogenesis Differentiation Kit; Gibco™; A1007001) for 7 days in CO₂ in cubator. Adipogenic differentiation medium was changed for every two days. At the end of 7 days of induction, cells were fixed with 4% paraformaldehyde for 10 minutes in room temperature and washed with DPBS twice. Cells were stained with 0.3 per cent of Oil Red ‘O’ (Sigma®; O1391) for 50 minutes in room temperature to detect the lipid droplets in the cell cytoplasm.

In primary culture of canine hair follicles bulge, round cells with clear cytoplasm was observed. On second day most of the cells were shown plastic adherence. The adhered cHFSCs showed small cytoplasmic processes surrounding round to oval nucleus with two or more nucleoli. The colonies became larger and united together forming a monolayer of cHFSC in the culture plate. Most of the colonies looked like cobblestone on appearance in the seventh day (Fig 1). Then cHFSCs were subjected to passage 1 (P1) (Fig 2) and passage 2 (P2) (Fig 3). The CD at primary culture was found to be 0.11±0.21×106 which remained the same to 0.11±0.24×106 at P1. The CD at P2 was higher than P1 and measured about 0.49±0.10×106. The PDT of cHFSCs in primary culture was observed as 4.36±0.02 hours in collagen coated culture plates. The PDT slightly increased in P1 as 4.38±0.02 hours than primary culture. The PDT of P2 was found to be 4.29±0.06 hours which was lesser than P1 culture.



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Immunocytochemistry
 
The cultured cells of the hair follicle were immunostained with Anti-Cytokeratin 15 and DAPI on day seven of post incubation. The positive stem cells were confirmed by observing intra cytoplasmic green fluorescence and intra nuclear blue fluorescence (Fig 4).
 
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Acridine orange staining
 
The viability of cells during culture was assessed by staining the primary cultured cells and P1 cells with Acridine orange. Viable cells were observed with emission of green fluorescence. The cells which were not viable appeared to emit light orange to red fluorescence in nucleus and yellow fluorescence cytoplasm. In primary culture, about 70 per cent of cHFSCs emitted green fluorescence (Fig 5).

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Adipogenic differentiated cHFSCs
 
After incubation of cHFSCs in adipogenic media, on the second day of induction, the cell morphology changed from cobblestone appearance to long spindle shape cells with long cytoplasmic process (Fig 6). On day three, fat droplets were observed in the cytoplasm of differentiated cells and became rounded in appearance. On day seven of post induction with adipogenic media, cytoplasm of cells contained clusters of fat droplets (Fig 7) which were positive to Oil red ‘O’ staining (Fig 8).

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In this study, cultured cHFSCs gave rise to macroscopic colonies within six days after initial seeding and colonies were homogenously made up of small flat cells near the periphery and the interior of the colony was stratified and consisted of large flat cells as described by Barrandon and Greens in human in 1987. Cells were closely aligned and gave cobblestone morphology which was similar to the results of goat and human hair follicle stem cell cultures respectively (Zhang et al., 2006; Hilmi et al., 2013).  

In the present study, only flat keratinocyte type of cells were isolated from canine hair follicles whereas de Castro reported that mixed population of cells, mostly round and fusiform shaped cells were isolated from canine fetal and adult skin (de Castro et al., 2018).In the cell viability assay, the dead cells were observed as yellow to red in fluorescence and viable cells fluoresced green in colour as reported by Bank (1988). Sieber-Blum et al., 2004 reported that the initial doubling time of mouse HFSCs was approximately six hours and the PDT for human HFSCs cultured in uncoated plate with CnT07 medium was 21.48±0.44 hours and in KSFM coated plates, it was 30.73±0.75 hours (Hilmi et al., 2013) which was reduced to 4.36±0.02 hours in primary culture of cHFSCs cultured in collagen coated plates and PDT was decreased in P2 level and calculated as 4.29±0.06 hours in the present study.   

The expression of CK15 transcription factor and DAPI was demonstrated as intra cytoplasmic green fluorescence and intranuclear blue fluorescence respectively in cultured cHFSCs from 7-14 days of culture as in gHFSC (He et al., 2016) and in human HFSCs, positive expression was noticed along with CD200, CD34, K14, K15, CD271 and K1 markers (Hilmi et al., 2013; Inoue et al., 2009). Shen also performed immunofluorescence detection for K19 and b1-integrin in human HFSCs (Shen et al., 2017). The hair follicle pluripotent nestin-driven GFP stem cells were positive for the stem cell marker CD34 but negative for CK15 and b-III tubulinin mice (Amoh et al., 2005).

Stem cells from dermal papilla of human hair follicles exhibited mesenchymal stem cell (MSC) immunophenotype and differentiated into all mesenchymal lineages and they were termed as human hair follicle derived mesenchymal stem cells (hHF-MSCs) (Liu et al., 2010). Adipogenesis property had decreased significantly in over time (Bajpai et al., 2012) Interestingly, loss of adipogenic differentiation potential was prevented by the adhesion of human Bone marrow mesenchymal stem cells to denatured type I collagen, suggesting a critical role of extracellular matrix in the maintenance of stem cell multipotency (Mauney et al., 2005). Adipogenic differentiation of canine hair follicle bulge stem cells has not been reported yet. In-vitro adipogenesis from cHFSCs was evident by aggregation of lipid rich droplets with cluster of differentiated cells stained positive by Oil red ‘O’ cytochemical staining after seven days of induction period. Review of earlier reports have evinced that the adipogenic differentiation of HFSCs from canine bulge cells was not proved in any species so far. Further validation of adipogenic differentiation of cHFSCs through qPCR is needed to confirm the differentiation property which was not performed in this study.

We thank the Dean, Madras Veterinary College (MVC), TANUVAS, Chennai, for providing necessary funding to carry out the research. We acknowledge the Department of Surgery and Radiology, MVC, for providing skin samples and Centre for Stem Cell Research and Regenerative Medicine, MVC, for the lab facilities and guidance.