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.
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).
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).
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).
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.
