Long-term expansion and differentiation of adult murine epidermal stem cells in 3D organoid cultures

Boonekamp, Kim E.; Kretzschmar, Kai; Wiener, Dominique J; Asra, Priyanca; Derakhshan, Sepideh; Puschhof, Jens; López‐Iglesias, Carmen; Peters, Peter J.; Basak, Onur; Clevers, Hans

doi:10.1073/pnas.1715272116

Cited by 64 publications

(69 citation statements)

References 58 publications

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“…It appears that Epi-SCs preserve their memory to form a stratified layer resembling the epidermis in culture and after transplantation into excisional wounds. This ability could be well preserved in the cells even after a longterm culture expansion [32]. In the present study, we found that Epi-SCs could migrate to form a distinctive epidermis-like layer over the "dermis" when cultured in mixture with SKPs in Matrigel.…”

Section: Discussionsupporting

confidence: 58%

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

et al. 2020

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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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Section: Discussionsupporting

confidence: 58%

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

et al. 2020

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“…Human skin tissue samples were obtained from Chinese PLA General Hospital, and ethical approval and informed consent were obtained. The interfollicular epidermal stem cells were isolated as described [ 19 , 20 ] with some modifications. Briefly, subcutaneous tissues and fat were removed and the tissues were digested with Dispase II (2.5 mg/mL, Cat #17150041, Gibco) at 37 °C for 2 h to obtain the epidermal sheets.…”

Section: Methodsmentioning

confidence: 99%

Both Wnt signaling and epidermal stem cell-derived extracellular vesicles are involved in epidermal cell growth

Leng

Lv³

et al. 2020

Stem Cell Res Ther

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Millions suffer from skin diseases. Functional interfollicular epidermal stem cells are needed in skin therapy or drug screening in vitro. We obtained functional interfollicular epidermal stem cells with intact stemness and cell junctions by treating them with Wnt3a. Moreover, epidermal stem cell-derived extracellular vesicles were useful in epidermal cell growth. Finally, functional epidermal 3D organoids with polarity were cultured using Wnt3a and the supernatant derived from interfollicular epidermal stem cells and fresh medium in a 1:1 ratio. These results provide novel directions for the improvement of skin organoids and their potential in clinical application.

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“…To avoid this potential risk, chemically defined culture media without animal-derived ingredients have been developed and are commercially available [24]. These feeder-and serum-free media [25] enable clonal growth and serial culture of EPSCs, but it is still unclear whether they can maintain EPSCs for long periods ex vivo.…”

Section: Expansion Of Epscs In Vitromentioning

confidence: 99%

Progress in studies of epidermal stem cells and their application in skin tissue engineering

Yang

Zhao

et al. 2020

Stem Cell Res Ther

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The epidermis, which is the outermost layer of mammalian skin, provides an essential barrier that is essential for maintenance of life. The epidermis is a stratified epithelium, which is maintained by the proliferation of epidermal stem cells (EPSCs) at the basal layer of the epidermis. As a unique cell population characterized by self-renewal and differentiation capabilities, EPSCs ensure the maintenance of adult skin homeostasis and participate in repair of the epidermis after injury. Recently, the utilization of EPSCs for wound healing and tissue regeneration has been attracting increased attention from researchers. In addition, the advances in tissue engineering have increased the interest in applying EPSCs in tissue-engineered scaffolds to further reconstitute injured tissues. In this review, we introduce research developments related to EPSCs, including methods recently used in the culture and enrichment of EPSCs, as well as advanced tools to study EPSCs. The function and mechanism of the EPSC-dermal units in the development and homeostasis of the skin are also summarized. Finally, the potential applications of EPSCs in skin tissue engineering are discussed.

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Long-term expansion and differentiation of adult murine epidermal stem cells in 3D organoid cultures

Cited by 64 publications

References 58 publications

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

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

Both Wnt signaling and epidermal stem cell-derived extracellular vesicles are involved in epidermal cell growth

Progress in studies of epidermal stem cells and their application in skin tissue engineering

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