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Clonal growth of dermal papilla cells in hydrogels reveals intrinsic differences between Sox2-positive and -negative cells in vitro and in vivo.

Driskell RR, Juneja VR, Connelly JT, Kretzschmar K, Tan DW, Watt FM - J. Invest. Dermatol. (2011)

Bottom Line: In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2- DP are associated with zigzag (ZZ) hairs.Nevertheless, spheres formed by CD133- cells did not efficiently support hair follicle formation in skin reconstitution assays.Hair type did not correlate with sphere size.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK.

ABSTRACT
In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2- DP are associated with zigzag (ZZ) hairs. We describe a three-dimensional hydrogel culture system that supports clonal growth of CD133+Sox2+, CD133+Sox2-, and CD133-Sox2- (non-DP) neonatal dermal cells. All three cell populations formed spheres that expressed the DP markers alkaline phosphatase, α8 integrin, and CD133. Nevertheless, spheres formed by CD133- cells did not efficiently support hair follicle formation in skin reconstitution assays. In the presence of freshly isolated P2 dermal cells, CD133+Sox2+ and CD133+Sox2- spheres contributed to the DP of both AA and ZZ hairs. Hair type did not correlate with sphere size. Sox2 expression was maintained in culture, but not induced significantly in Sox2- cells in vitro or in vivo, suggesting that Sox2+ cells are a distinct cellular lineage. Although Sox2+ cells were least efficient at forming spheres, they had the greatest ability to contribute to DP and non-DP dermis in reconstituted skin. As the culture system supports clonal growth of DP cells and maintenance of distinct DP cell types, it will be useful for further analysis of intrinsic and extrinsic signals controlling DP function.

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Sox2-positive dermal spheres contribute to dermal papilla (DP) and non-DP dermis. (a) Schematic representation of hair reconstitution assay using wild-type dermal ‘helper' cells. (b–d) Macroscopic appearance of grafts after 4 weeks. (e–g) Dermal side of grafts viewed under a fluorescence-dissecting microscope. Note that green fluorescent protein (GFP)-positive DP cells are only detected in g. (h–m) Thick cryosections of graft sites immunolabeled for GFP (h–j) or dsRed (k–m) with 4′-6-diamidino-2-phenylindole counterstain (blue). (k–m) dsRed-positive DP cells are indicated with arrows and dsRed-positive non-DP dermal cells with an asterisk. Bars=150 μm. (n) Percentage of DP derived from hydrogel cultures. Values are significantly higher for CD133+ than CD133− cultures (***P<0.001). (o) Number of hairs of each type formed per graft. Means±SEM are indicated. Values that differed significantly from CD133+GFP− are shown (*P<0.05; **P<0.01). (n, o) N=number of grafts analyzed.
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fig6: Sox2-positive dermal spheres contribute to dermal papilla (DP) and non-DP dermis. (a) Schematic representation of hair reconstitution assay using wild-type dermal ‘helper' cells. (b–d) Macroscopic appearance of grafts after 4 weeks. (e–g) Dermal side of grafts viewed under a fluorescence-dissecting microscope. Note that green fluorescent protein (GFP)-positive DP cells are only detected in g. (h–m) Thick cryosections of graft sites immunolabeled for GFP (h–j) or dsRed (k–m) with 4′-6-diamidino-2-phenylindole counterstain (blue). (k–m) dsRed-positive DP cells are indicated with arrows and dsRed-positive non-DP dermal cells with an asterisk. Bars=150 μm. (n) Percentage of DP derived from hydrogel cultures. Values are significantly higher for CD133+ than CD133− cultures (***P<0.001). (o) Number of hairs of each type formed per graft. Means±SEM are indicated. Values that differed significantly from CD133+GFP− are shown (*P<0.05; **P<0.01). (n, o) N=number of grafts analyzed.

Mentions: We next compared the hair-forming capacity of cultured CD133−Sox2−, CD133+Sox2−, and CD133+Sox2+ dermal cells by grafting disaggregated spheres in combination with an excess of uncultured, unfractionated P2 dermal cells (Figure 6a). For these experiments, dermal cells were isolated from Sox2eGFP/CAG-dsRed mice, labeled with anti-CD133, sorted, and cultured in AmnioMax hydrogels for 2 weeks. The helper P2 dermal cells and the epidermal cells were from wild-type mice and did not express GFP or dsRed (Figure 6a). Three grafts were performed with each type of sphere (CD133−Sox2−, CD133+Sox2−, and CD133+Sox2+).


Clonal growth of dermal papilla cells in hydrogels reveals intrinsic differences between Sox2-positive and -negative cells in vitro and in vivo.

Driskell RR, Juneja VR, Connelly JT, Kretzschmar K, Tan DW, Watt FM - J. Invest. Dermatol. (2011)

Sox2-positive dermal spheres contribute to dermal papilla (DP) and non-DP dermis. (a) Schematic representation of hair reconstitution assay using wild-type dermal ‘helper' cells. (b–d) Macroscopic appearance of grafts after 4 weeks. (e–g) Dermal side of grafts viewed under a fluorescence-dissecting microscope. Note that green fluorescent protein (GFP)-positive DP cells are only detected in g. (h–m) Thick cryosections of graft sites immunolabeled for GFP (h–j) or dsRed (k–m) with 4′-6-diamidino-2-phenylindole counterstain (blue). (k–m) dsRed-positive DP cells are indicated with arrows and dsRed-positive non-DP dermal cells with an asterisk. Bars=150 μm. (n) Percentage of DP derived from hydrogel cultures. Values are significantly higher for CD133+ than CD133− cultures (***P<0.001). (o) Number of hairs of each type formed per graft. Means±SEM are indicated. Values that differed significantly from CD133+GFP− are shown (*P<0.05; **P<0.01). (n, o) N=number of grafts analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3306894&req=5

fig6: Sox2-positive dermal spheres contribute to dermal papilla (DP) and non-DP dermis. (a) Schematic representation of hair reconstitution assay using wild-type dermal ‘helper' cells. (b–d) Macroscopic appearance of grafts after 4 weeks. (e–g) Dermal side of grafts viewed under a fluorescence-dissecting microscope. Note that green fluorescent protein (GFP)-positive DP cells are only detected in g. (h–m) Thick cryosections of graft sites immunolabeled for GFP (h–j) or dsRed (k–m) with 4′-6-diamidino-2-phenylindole counterstain (blue). (k–m) dsRed-positive DP cells are indicated with arrows and dsRed-positive non-DP dermal cells with an asterisk. Bars=150 μm. (n) Percentage of DP derived from hydrogel cultures. Values are significantly higher for CD133+ than CD133− cultures (***P<0.001). (o) Number of hairs of each type formed per graft. Means±SEM are indicated. Values that differed significantly from CD133+GFP− are shown (*P<0.05; **P<0.01). (n, o) N=number of grafts analyzed.
Mentions: We next compared the hair-forming capacity of cultured CD133−Sox2−, CD133+Sox2−, and CD133+Sox2+ dermal cells by grafting disaggregated spheres in combination with an excess of uncultured, unfractionated P2 dermal cells (Figure 6a). For these experiments, dermal cells were isolated from Sox2eGFP/CAG-dsRed mice, labeled with anti-CD133, sorted, and cultured in AmnioMax hydrogels for 2 weeks. The helper P2 dermal cells and the epidermal cells were from wild-type mice and did not express GFP or dsRed (Figure 6a). Three grafts were performed with each type of sphere (CD133−Sox2−, CD133+Sox2−, and CD133+Sox2+).

Bottom Line: In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2- DP are associated with zigzag (ZZ) hairs.Nevertheless, spheres formed by CD133- cells did not efficiently support hair follicle formation in skin reconstitution assays.Hair type did not correlate with sphere size.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK.

ABSTRACT
In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2- DP are associated with zigzag (ZZ) hairs. We describe a three-dimensional hydrogel culture system that supports clonal growth of CD133+Sox2+, CD133+Sox2-, and CD133-Sox2- (non-DP) neonatal dermal cells. All three cell populations formed spheres that expressed the DP markers alkaline phosphatase, α8 integrin, and CD133. Nevertheless, spheres formed by CD133- cells did not efficiently support hair follicle formation in skin reconstitution assays. In the presence of freshly isolated P2 dermal cells, CD133+Sox2+ and CD133+Sox2- spheres contributed to the DP of both AA and ZZ hairs. Hair type did not correlate with sphere size. Sox2 expression was maintained in culture, but not induced significantly in Sox2- cells in vitro or in vivo, suggesting that Sox2+ cells are a distinct cellular lineage. Although Sox2+ cells were least efficient at forming spheres, they had the greatest ability to contribute to DP and non-DP dermis in reconstituted skin. As the culture system supports clonal growth of DP cells and maintenance of distinct DP cell types, it will be useful for further analysis of intrinsic and extrinsic signals controlling DP function.

Show MeSH
Related in: MedlinePlus