Investigation of characteristics of feather follicle stem cells and their regeneration potential

Abstract

Feather follicles have the extraordinary ability to regenerate and undergo molting cycles. Being tissue-specific stem cells, feather follicle stem cells (FFSCs) have a strong capacity for proliferation and are presumed to be progenitor cells for various epidermal organs. In order to characterize FFSCs and to understand how the feather epidermis and FFSCs produce such a reliable differentiation program resulting in the formation of complex feathers, We developed a culture scheme to select and expand FFSCs from chick feather follicles. FFSCs were examined with cell profiles, mutilpotential differentiation and immunocytochemical staining. FFSCs from a single clone were capable of self-renewal and proliferation. These cells expressed integrin β1, CD49c, cytokeratin 15 (K15), cytokeratin 19 (K19) and a neural-genic cell marker, nestin, but not a teminal differentiation-related keratinocyte marker, cytokeratin 10 (K10). FFSCs could trans-differentiate into adipocytes, neurocytes and keratinocytes. The formation of micro-feather like structures ex-vivo also revealed the potential of regeneration. These results demonstrate that FFSCs possess the properties of stem/progenitor cells and may therefore serve as a useful model for studying mechanisms of stem cell differentiation and their involvement in organ regeneration.

Keywords: Feather follicle stem cells; regeneration; trans-differentiation.

Figure 1

Cellular characteristics of FFSCs. Paraffin sections of feather follicle showed collar and dermal papilla, from which the putative FFSCs were isolated (A). Cells attached to the plate appear triangular or polygonal in morphology after culture for 150 min in the early selection scheme (B). Colonies with passage potential were intensively observed during the culture(C). Later, the cells at subconfluence showed a typical cobblestone shape (D). The growth curves for cells selected in the early and later selection schemes at passages 1, 3 and 10 are showing in (I) and (J). The two growth trends are similar but the growth potential is significantly different (P<0.05). Cell cycle analysis showed that in the early scheme, cells in G0/G1 phases were more numerous than cells in other phases (K). FFSCs were confirmed by the immunocytochemicall staining with such cellular markers as integrin β1 (E), CD49c (F), K19 (G) and K15 (H).

Figure 2

Fluorescence double staining for K15 and nestin at the different stage of neurogenesis culture. Both K15-positive and nesting positive cells appeared in the initial FFSCs culture (A1-B1). The similar situation was detected after pre-induction for 24 hr. When the induction lasted for 5 h, cells expressed no K15 (A5) but express nestin (B5). Images of C1-C3 show DAPI staining for the nuclei.

Figure 3

Mutilineage differentiation of FFSCs and 3-dimention potential. During neurogenic differentiation, FFSCs derived cells appeared as progenitor cells with neural-like characteristics and neurocytes gradually. The cells appeared negative to nestin (A), but positive to GFAP (B) and NSE (C) at the stage of 24 hr induction. Induced cells were analyzed by RT-PCR for nestin mRNA expression during induction for 0–24 h; β-actin served as an internal control (I). The relationship between fluorescence intensity and induction time showed that nestin expression decreased gradually as induction continued (J). In adipogenesis culture, cells were induced for 5 days and typical mature fat morphology and lipid vacuoles showing strong lipid refraction were observed. After a double staining with Oil Red O and K15, lipid vacuoles showed orange or red but no blue color for K15, indicating that cells were differentiated into adipocytes (D). Relationship between triglyceride content and days following initial adipogenic induction were showed in (K). By RT-PCR, the mRNA expression of PPARγ was not detectable in initial culture of FFSCs (L, lane 1) but in FFSCs-deried adipocytes (L, lane 2). In the assay of FFSCs differentiated into keratinocytes, cells changed from cobblestone shape to ovate one after induction for 2 days. Stratified epithelium and desmosomes were observed after 5 days. Induced cells were intensively positive to K10 (E). The formation of micro-structures was observed in cells obtained from the later selection scheme, not the early selection scheme. These structures such as cell clusters (F), hierarchical branches and micro-feather like structures with hierarchical branches (G) and multi-layered transformations of a keratinocyte sheet appeared (H).