Cultured Skin-Based Stem Cells Regenerate Hair Follicles and Sebaceous Glands


It has been over ten years since the development of cultured skin substitutes or CSSs. CSSs consist of cultured epidermis from the patients and dermal fibroblasts that can form a good epidermal layer. However, CSSs do not form hair follicles or sebaceous glands which makes for mighty dry skin. Therefore, it is highly preferable to make at skin substitute that can form such epidermal appendages.

Fortunately the skin possesses several types of stem cells for use in regenerative medicine. Epidermal stem cells (Epi-SCs) in the basal layer of the epidermis that constantly provide new cells to the epidermis (the uppermost layer of the skin). Unfortunately, adult Epi-SCs cannot form hair follicles, but they can if they are combined with embryonic or newborn dermal cells. Dermal papilla (DP) cells can induce hair follicles, but the availability of DP cells has greatly limited work with them. Adult dermal skin also contains multipotent SKPs or skin-derived precursors. Injection of SKPs underneath the skin of mice, leads to the induction of new hair follicles (Biernaskie et al., 2009, Cell Stem Cell 5:610-623). This suggests that SKPs might be applicable in a clinical setting to induce hair follicles in cultured skin substitutes.

A new paper by Yaojiong Wu and coworker from the Shenzhen Key Laboratory of Health Sciences and Technology, in collaboration with Edward E. Tredget from University College Dublin has successfully induced the growth of hair follicles and sebaceous glands in a cultured skin substitute. They combined cultured Epi-SCs and SKPs, from mice and humans, and embedded them into a hydrogel. These stem cell-embedded hydrogels were then implanted into immunodeficient mice with substantial skin wounds.

The results were remarkable. The implants able to induce the formation of new epidermis with hair follicles. Furthermore, when Epi-SCs and SKPs taken from human scalps were used in these experiments, they worked just as well as those taken from mice.

Hair neogenesis with cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs). (A): Putative epidermal stem cells residing in the basal layer of neonatal mouse epidermis expressed CD49f (red) in immunofluorescence stain, and mature keratinocytes in the top layers of the epidermis expressed cytokeratin (CK)6 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole. (B–E): Cultured Epi-SCs derived from neonatal mice (B) were positive for CD49f (C) and CK15 (D) in immunofluorescence stain; fluorescence-activated cell sorting analysis of the Epi-SCs indicated high levels of surface CD29 and CD49f (E). (F): The expression level of CD49f decreased progressively upon successive passages (P) in culture as determined by immunofluorescence analysis (in relation to the fluorescence intensity of P0 cells). Triple wells were used for each of the above experiments, and each experiment was repeated three times with similar results (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (G): Hair genesis of cultured Epi-SCs in different passages. Cultured Epi-SCs derived from neonatal mice in different passages (P0 to P5) were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal dermal cells (fresh D) in Matrigel; dermal cells alone or freshly isolated neonatal epidermal cells plus dermal cells (fresh E+D) were used as controls. Hair shafts generated 20 days posttransplant were counted (n = 6; ∗, p < .05; ∗∗∗, p < .001). (H–J): SKPs derived from neonatal mice in spheroid culture (H) expressed nestin, fibronectin (I), and BMP6 (J) in immunofluorescence analysis. (K): Hair genesis of SKPs in different passages. SKPs in P0 to P5 were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal mouse epidermal cells (fresh E), and freshly isolated neonatal mouse epidermal cells alone or in combination with freshly isolated neonatal mouse dermal cells (fresh E+D) were used as controls. Twenty days posttransplant, hairs generated were counted (n = 6; ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (L-N): Cultured Epi-SCs and SKPs in hair genesis. Combinations of cultured neonatal mouse Epi-SCs (P0 to P3) and SKPs (P0 to P3) were engrafted into excisional wounds in nude mice, and the number of hairs generated were counted 20 days posttransplant (n = 3, ∗, p < .05). (L). A representative image of hairs generated 20 days after a transplantation of P1 Epi-SCs and SKPs (M). Immunofluorescence analysis of the skin tissue with hair genesis showed densely populated hair follicles and sebaceous glands (N). Scale bars = 50 μm. Abbreviations: BM, basement membrane; BMP6, bone morphogenetic protein 6; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; Derm, dermis; Epi, epidermis; Epi-SC, epidermal stem cells; FITC, fluorescein isothiocyanate; fresh D, freshly isolated neonatal dermal cells; fresh D+E, freshly isolated neonatal epidermal cells plus dermal cells; HF, hair follicle; HS, hair shafts; P, passage; PE, phycoerythrin.
Hair neogenesis with cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs). (A): Putative epidermal stem cells residing in the basal layer of neonatal mouse epidermis expressed CD49f (red) in immunofluorescence stain, and mature keratinocytes in the top layers of the epidermis expressed cytokeratin (CK)6 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole. (B–E): Cultured Epi-SCs derived from neonatal mice (B) were positive for CD49f (C) and CK15 (D) in immunofluorescence stain; fluorescence-activated cell sorting analysis of the Epi-SCs indicated high levels of surface CD29 and CD49f (E). (F): The expression level of CD49f decreased progressively upon successive passages (P) in culture as determined by immunofluorescence analysis (in relation to the fluorescence intensity of P0 cells). Triple wells were used for each of the above experiments, and each experiment was repeated three times with similar results (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (G): Hair genesis of cultured Epi-SCs in different passages. Cultured Epi-SCs derived from neonatal mice in different passages (P0 to P5) were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal dermal cells (fresh D) in Matrigel; dermal cells alone or freshly isolated neonatal epidermal cells plus dermal cells (fresh E+D) were used as controls. Hair shafts generated 20 days posttransplant were counted (n = 6; ∗, p < .05; ∗∗∗, p < .001). (H–J): SKPs derived from neonatal mice in spheroid culture (H) expressed nestin, fibronectin (I), and BMP6 (J) in immunofluorescence analysis. (K): Hair genesis of SKPs in different passages. SKPs in P0 to P5 were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal mouse epidermal cells (fresh E), and freshly isolated neonatal mouse epidermal cells alone or in combination with freshly isolated neonatal mouse dermal cells (fresh E+D) were used as controls. Twenty days posttransplant, hairs generated were counted (n = 6; ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (L-N): Cultured Epi-SCs and SKPs in hair genesis. Combinations of cultured neonatal mouse Epi-SCs (P0 to P3) and SKPs (P0 to P3) were engrafted into excisional wounds in nude mice, and the number of hairs generated were counted 20 days posttransplant (n = 3, ∗, p < .05). (L). A representative image of hairs generated 20 days after a transplantation of P1 Epi-SCs and SKPs (M). Immunofluorescence analysis of the skin tissue with hair genesis showed densely populated hair follicles and sebaceous glands (N). Scale bars = 50 μm. Abbreviations: BM, basement membrane; BMP6, bone morphogenetic protein 6; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; Derm, dermis; Epi, epidermis; Epi-SC, epidermal stem cells; FITC, fluorescein isothiocyanate; fresh D, freshly isolated neonatal dermal cells; fresh D+E, freshly isolated neonatal epidermal cells plus dermal cells; HF, hair follicle; HS, hair shafts; P, passage; PE, phycoerythrin.

Additionally, when the ability of Epi-SCs to differentiate into sebaceous glands was examined, Wu and others showed that Epi-SCs can form the precursors to sebaceous glands, sebocytes. Additionally, the oils secreted by these Epi-SC-derived sebocytes were chemically similar to sebaceous glands from native skin.

Thus, a combination of Epi-SCs and SKPs from human or mouse skin were sufficient to generate newly formed hair follicles and functional sebaceous glands. These results provide knowledge that is potentially transferable to clinical applications for regenerating damaged skin.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).