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Cited by 152Hair Growth and DisordersSkin and Cellular Biology Researchdental development and anomalies

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PI3K/Akt signaling pathway is essential for de novo hair follicle regeneration

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Chen, Yu, Fan, Zhimeng, Wang, Xiaoxiao, Mo, Miaohua, Zeng, Shu Bin, Xu, Ren-He, Wang, Xusheng, Wu, Yaojiong·2020·Stem Cell Research & Therapy
DOI 10.1186/s13287-020-01650-6PMC PMC7118821
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Abstract

Background: Cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs) were capable of reconstituting functional hair follicles after implantation, while the signaling pathways that regulate neogenic hair follicle formation are poorly investigated. In this study, we aimed to understand the interactions between Epi-SCs and SKPs during skin organoid formation and to uncover key signal pathways crucial for de novo hair follicle regeneration. Methods: To track their fate after transplantation, Epi-SCs derived from neonatal C57BL/6 mice were labeled with tdTomato, and SKPs were isolated from neonatal C57BL/6/GFP mice. A mixture of Epi-SCs-tdTomato and SKPs-EGFP in Matrigel was observed under two-photon microscope in culture and after implantation into excisional wounds in nude mice, to observe dynamic migrations of the cells during hair follicle morphogenesis. Signaling communications between the two cell populations were examined by RNA-Seq analysis. Potential signaling pathways revealed by the analysis were validated by targeting the pathways using specific inhibitors to observe a functional loss in de novo hair follicle formation. Results: Two-photon microscopy analysis indicated that when Epi-SCs and SKPs were mixed in Matrigel and cultured, they underwent dynamic migrations resulting in the formation of a bilayer skin-like structure (skin organoid), where Epi-SCs positioned themselves in the outer layer; when the mixture of Epi-SCs and SKPs was grafted into excisional wounds in nude mice, a bilayer structure resembling the epidermis and the dermis formed at the 5th day, and de novo hair follicles generated subsequently. RNA-Seq analysis of the two cell types after incubation in mixture revealed dramatic alterations in gene transcriptome, where PI3K-Akt signaling pathway in Epi-SCs was significantly upregulated; meanwhile, elevated expressions of several growth factors and cytokine potentially activating PI3K were found in SKPs, suggesting active reciprocal communications between them. In addition, inhibition of PI3K or Akt by specific inhibitors markedly suppressed the hair follicle regeneration mediated by Epi-SCs and SKPs. Conclusions: Our data indicate that the PI3K-Akt signaling pathway plays a crucial role in de novo hair follicle regeneration, and the finding may suggest potential therapeutic applications in enhancing hair regeneration.

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Fig. 1 A schematic diagram of Epi-SCs and SKP transplantation for hair follicle regeneration. Dorsal skin tissue in full thickness was collected from neonatal C57 mice (a), which was cut into pieces (b). The tissue was separated into the epidermis and dermis after treatment with dispase II (c, d). Epidermal stem cells (Epi-SCs) were derived from the epidermis as described in the “Methods” section, which grew in monolayer (e) and expressed CD49f as detected by immunofluorescence staining (red, g). Skin-derived precursors (SKPs) were derived from the dermis as described in the “Methods” section, which were grown in spheroids and expressed nestin as detected by immunofluorescence staining (red, h). Full thickness excisional skin wounds were prepared in nu/nu mice (i), and a mixture of Epi-SCs and SKPs in Matrigel was implanted into the wound, which resulted in the growth of black hairs (j). Scale bar, 50 μm
Fig. 1 A schematic diagram of Epi-SCs and SKP transplantation for hair follicle regeneration. Dorsal skin tissue in full thickness was collected from neonatal C57 mice (a), which was cut into pieces (b). The tissue was separated into the epidermis and dermis after treatment with dispase II (c, d). Epidermal stem cells (Epi-SCs) were derived from the epidermis as described in the “Methods” section, which grew in monolayer (e) and expressed CD49f as detected by immunofluorescence staining (red, g). Skin-derived precursors (SKPs) were derived from the dermis as described in the “Methods” section, which were grown in spheroids and expressed nestin as detected by immunofluorescence staining (red, h). Full thickness excisional skin wounds were prepared in nu/nu mice (i), and a mixture of Epi-SCs and SKPs in Matrigel was implanted into the wound, which resulted in the growth of black hairs (j). Scale bar, 50 μmCC BY · PubMed Central
Fig. 2 Fates of Epi-SCs and SKPs in mixture in vitro and in vivo. a–f Epi-SCs and SKPs in skin organoid formation. Epi-SCs labeled with tdTomato (red) were cultured in monolayer (a) and SKPs derived from C57-EGFP mice were grown in spheroids (b). Single cells of the above were mixed evenly in Matrigel (c, d) to form a sphere, which was incubated at 37 °C for 24 h. Cross-sections of the sphere showed that the cells were repopulated into two compartments, with the Epi-SCs-tdTomato in the outer layer (red) and the SKPs-EGFP (green) in the inner compartment (e, f), resembling the structure of bilayer skin. g–r Fates of Epi-SCs and SKPs in vivo. A mixture of single Epi-SCs-tdTomato and SKPs-EGFP in Matrigel was implanted into an excisional wound in a nude mouse, and the graft was observed under two-photon microscope. In the first 3 days, Epi-SCs aggregated forming spheres (red, g and h). By day 5, Epi-SCs migrated upward and formed an epidermis-like layer over SKPs (i, j). Some Epi-SCs in the layer then moved downward into the SKPs forming a primary structure of the hair follicle by 12 days; images of graft surface (k), horizontal section (l), and vertical section (m) were shown. n–q Tissue sections of the wound at 14 days post transplantation showed that the Epi-SCs and SKPs formed de novo skin structures, where the DPs and dermal cells were derived from the GFP-expressing SKPs (n, q); the epidermis and the trunk of the hair follicle were formed by the Epi-SCs-tdTomato (o, q). r, s At the interface of DP and follicle germ was the matrix (r, s). HF, hair follicle; Epi, epidermis; DP, dermal papilla. Scale bar, 100 μm
Fig. 2 Fates of Epi-SCs and SKPs in mixture in vitro and in vivo. a–f Epi-SCs and SKPs in skin organoid formation. Epi-SCs labeled with tdTomato (red) were cultured in monolayer (a) and SKPs derived from C57-EGFP mice were grown in spheroids (b). Single cells of the above were mixed evenly in Matrigel (c, d) to form a sphere, which was incubated at 37 °C for 24 h. Cross-sections of the sphere showed that the cells were repopulated into two compartments, with the Epi-SCs-tdTomato in the outer layer (red) and the SKPs-EGFP (green) in the inner compartment (e, f), resembling the structure of bilayer skin. g–r Fates of Epi-SCs and SKPs in vivo. A mixture of single Epi-SCs-tdTomato and SKPs-EGFP in Matrigel was implanted into an excisional wound in a nude mouse, and the graft was observed under two-photon microscope. In the first 3 days, Epi-SCs aggregated forming spheres (red, g and h). By day 5, Epi-SCs migrated upward and formed an epidermis-like layer over SKPs (i, j). Some Epi-SCs in the layer then moved downward into the SKPs forming a primary structure of the hair follicle by 12 days; images of graft surface (k), horizontal section (l), and vertical section (m) were shown. n–q Tissue sections of the wound at 14 days post transplantation showed that the Epi-SCs and SKPs formed de novo skin structures, where the DPs and dermal cells were derived from the GFP-expressing SKPs (n, q); the epidermis and the trunk of the hair follicle were formed by the Epi-SCs-tdTomato (o, q). r, s At the interface of DP and follicle germ was the matrix (r, s). HF, hair follicle; Epi, epidermis; DP, dermal papilla. Scale bar, 100 μmCC BY · PubMed Central
Fig. 3 RNA-Seq of Epi-SC and SKPs. a A schematic diagram depicting sample preparation for RNA-Seq. Total RNA was extracted from Epi-SCs-tdTomato and SKPs-EGFP which were cultured separately or in mixture in Matrigel for 24 h. Cells were recovered from the matrix and separated by cell sorting using a flow cytometer. Total RNA was extracted from the cells and subjected to RNA-Seq analysis. b Transcriptome shows different gene transcriptional patterns of Epi-SCs and SKPs when cultured alone or in mixture (x). c, d KEGG analysis revealed active signals in Epi-SCs and SKPs when cultured in mixture, among them there was PI3K/Akt signal
Fig. 3 RNA-Seq of Epi-SC and SKPs. a A schematic diagram depicting sample preparation for RNA-Seq. Total RNA was extracted from Epi-SCs-tdTomato and SKPs-EGFP which were cultured separately or in mixture in Matrigel for 24 h. Cells were recovered from the matrix and separated by cell sorting using a flow cytometer. Total RNA was extracted from the cells and subjected to RNA-Seq analysis. b Transcriptome shows different gene transcriptional patterns of Epi-SCs and SKPs when cultured alone or in mixture (x). c, d KEGG analysis revealed active signals in Epi-SCs and SKPs when cultured in mixture, among them there was PI3K/Akt signalCC BY · PubMed Central
Fig. 4 Analysis of PI3K/Akt inhibition on hair follicle regeneration. a Hair follicle reconstitution assay showed that addition of Periforsine or LY294002 to Epi-SCs and SKPs mixtures before transplantation inhibited the formation of neogenic hair follicles after implantation into excisional wounds in nude mice. b HE staining of 21-day post-grafted tissue sections with the absence (control) or presence of Perifosine or LY294002 in the graft. c Immunofluorescence staining of the wound tissue sections for the expression of Keratin (K) 14 and K1. Scale bars, 50 μm
Fig. 4 Analysis of PI3K/Akt inhibition on hair follicle regeneration. a Hair follicle reconstitution assay showed that addition of Periforsine or LY294002 to Epi-SCs and SKPs mixtures before transplantation inhibited the formation of neogenic hair follicles after implantation into excisional wounds in nude mice. b HE staining of 21-day post-grafted tissue sections with the absence (control) or presence of Perifosine or LY294002 in the graft. c Immunofluorescence staining of the wound tissue sections for the expression of Keratin (K) 14 and K1. Scale bars, 50 μmCC BY · PubMed Central

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Metadata

Authors
Chen, Y., Fan, Z., Wang, X., Mo, M., Zeng, S. B., Xu, R.-H., Wang, X., & Wu, Y.
Affiliations
  • Tsinghua Shenzhen International Graduate School (CN)
  • Sun Yat-sen University (CN)
  • Tsinghua–Berkeley Shenzhen Institute (CN)
  • University of Macau (MO)
Corresponding author
  • Wang, Xusheng
  • Wu, Yaojiong
ORCID
Venue
Stem Cell Research & Therapy
Publisher
Springer Science and Business Media LLC
Published
2020-04-03
Volume
11
Issue
1
Pages
144-144
ISSN
1757-6512 (ISSN-L)
DOI
10.1186/s13287-020-01650-6
PMC
PMC7118821
OpenAlex
W3014739274
Cited by
130
References
55
Citations by year
2026: 21 · 2025: 24 · 2024: 30 · 2023: 16 · 2022: 24 · 2021: 12 · 2020: 3
FWCI
19.06
Type
journal article
Language
en
Open access
Licence
Locations
  • Stem Cell Research & Therapy (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Natural Science Foundation of China (No. 31571429, 31961160702)
  • National Key R&D Program of China (2017YFC1103304)
  • Shenzhen Science and Technology Innovation Committee (JCYJ20170818093935581, JCYJ20180307123901314)
  • State Key Laboratory of Chemical Oncogenomics fund
Topics
  • Health Sciences › Medicine › Dermatology › Hair Growth and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Skin and Cellular Biology Research
  • Health Sciences › Dentistry › General Dentistry › dental development and anomalies
Keywords
PI3K/Akt signaling, epidermal stem cells, skin-derived precursors, hair follicle morphogenesis, cell migration, wound healing
MeSH terms
Animals, Cells, Cultured, Mice, Inbred C57BL, Mice, Nude, Signal Transduction, Hair Follicle, Phosphatidylinositol 3-Kinases, Proto-Oncogene Proteins c-akt, Mice

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