The empowering influence of air-liquid interface culture on skin organoid hair follicle development

Abstract

Background: Rodent models have been widely used to investigate skin development, but do not account for significant differences in composition compared to human skin. On the other hand, two-dimensional and three-dimensional engineered skin models still lack the complex features of human skin such as appendages and pigmentation. Recently, hair follicle containing skin organoids (SKOs) with a stratified epidermis, and dermis layer have been generated as floating spheres from human-induced pluripotent stem cells (hiPSCs). Methods: The current study aims to investigate the generation of hiPSCs-derived SKOs using an air-liquid interface (ALI) model on transwell membranes (T-SKOs) and compares their development with conventional floating culture in low-attachment plates (F-SKOs). Results: Mature SKOs containing an epidermis, dermis, and appendages are created in both T-SKO and F-SKO conditions. It was found that the hair follicles are smaller and shorter in the F-SKO compared with T-SKOs. Additionally, the ALI conditions contribute to enhanced hair follicle numbers than conventional floating culture. Conclusions: Together, this study demonstrates the significant influence of transwell culture on the morphogenesis of hair follicles within SKOs and highlights the potential for refinement of skin model engineering for advancing dermatology and skin research.

Keywords

Air-liquid interface / Dermal papilla / Pluripotent stem cell / Regenerative medicine / Skin appendages

References

[1] Zomer HD, Trentin AG. Skin wound healing in humans and mice: challenges in translational research. J Dermatol Sci. 2018; 90: 3-12. https://doi.org/10.1016/j.jdermsci.2017.12.009.

[2] Hosseini M, Brown J, Khosrotehrani K, Bayat A, Shafiee A. Skin biomechanics: a potential therapeutic intervention target to reduce scarring. Burns & trauma. 2022; 10: tkac036. https://doi.org/10.1093/burnst/tkac036.

[3] Xie Y, Rizzi SC, Dawson R, Lynam E, Richards S, Leavesley DI, et al. Development of a three-dimensional human skin equivalent wound model for investigating novel wound healing therapies. Tissue Engineering Part C: Methods. 2010; 16: 1111-23. https://doi.org/10.1089/ten.tec.2009.0725.

[4] Black AF, Bouez C, Perrier E, Schlotmann K, Chapuis F, Damour O. Optimization and characterization of an engineered human skin equivalent. Tissue Eng. 2005; 11: 723-33. https://doi.org/10.1089/ten.2005.11.723.

[5] Itoh M, Umegaki-Arao N, Guo Z, Liu L, Higgins CA, Christiano AM. Generation of 3D skin equivalents fully reconstituted from human induced pluripotent stem cells (iPSCs). PLoS One. 2013; 8: e77673. https://doi.org/10.1371/journal.pone.0077673.

[6] Amourizi FZ, Malek-Khatabi AZ, Zare-Dorabei R. Polymeric and composite-based microneedles in drug delivery: regenerative medicine, microbial infection therapy, and cancer treatment. Materials Chemistry Horizons. 2023; 2: 113-24.

[7] Hosseini M, Koehler KR, Shafiee A. Biofabrication of human skin with its appendages. Advanced healthcare materials. 2022; 11: e2201626. https://doi.org/10.1002/adhm.202201626.

[8] Lee J, Rabbani CC, Gao H, Steinhart MR, Woodruff BM, Pflum ZE, et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature. 2020; 582: 399-404. https://doi.org/10.1038/s41586-020-2352-3.

[9] Shafiee A, Sun J, Ahmed IA, Phua F, Rossi GR, Lin CY, et al. Development of physiologically relevant skin organoids from human induced pluripotent stem cells. Small. 2024; 20: e2304879. https://doi.org/10.1002/smll.202304879.

[10] Lee J, Koehler KR. Skin organoids: a new human model for developmental and translational research. Exp Dermatol. 2021; 30: 613-20. https://doi.org/10.1111/exd.14292.

[11] Wang X, Tredget EE, Wu Y. Dynamic signals for hair follicle development and regeneration. Stem Cells Dev. 2012; 21: 7-18. https://doi.org/10.1089/scd.2011.0230.

[12] Fuchs E. Scratching the surface of skin development. Nature. 2007; 445: 834-42. https://doi.org/10.1038/nature05659.

[13] Lee J, Böscke R, Tang P-C, Hartman BH, Heller S, Koehler KR. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 2018; 22: 242-54. https://doi.org/10.1016/j.celrep.2017.12.007.

[14] Pruniéras M, Régnier M, Woodley D. Methods for cultivation of keratinocytes with an air-liquid interface. J Invest Dermatol. 1983; 81: S28-33. https://doi.org/10.1111/1523-1747.ep12540324.

[15] Giandomenico SL, Mierau SB, Gibbons GM, Wenger LM, Masullo L, Sit T, et al. Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output. Nat Neurosci. 2019; 22: 669-79. https://doi.org/10.1038/s41593-019-0350-2.

[16] Jung S-y, You HJ, Kim M-J, Ko G, Lee S, Kang K-S. Wnt-activating human skin organoid model of atopic dermatitis induced by Staphylococcus aureus and its protective effects by Cutibacterium acnes. Iscience. 2022; 25: 105150. https://doi.org/10.1016/j.isci.2022.105150.

[17] Shafiee A, Patel J, Hutmacher DW, Fisk NM, Khosrotehrani K. Meso-endothelial bipotent progenitors from human placenta display distinct molecular and cellular identity. Stem cell reports. 2018; 10: 890-904. https://doi.org/10.1016/j.stemcr.2018.01.011.

[18] Ahmed I, Sun J, Brown J, Khosrotehrani K, Shafiee A. An optimized protocol for generating appendage-bearing skin organoids from human induced pluripotent stem cells. Biology Methods and Protocols. 2024; 9: bpae019. https://doi.org/10.1093/biomethods/bpae019.

[19] Qian X, Song H, Ming G-l. Brain organoids: advances, applications and challenges. Development. 2019; 146: dev166074. https://doi.org/10.1242/dev.166074.

[20] Duverger O, Morasso MI. Epidermal patterning and induction of different hair types during mouse embryonic development. Birth Defects Research Part C: Embryo Today: Reviews. 2009; 87: 263-72. https://doi.org/10.1002/bdrc.20158.

[21] Driskell RR, Giangreco A, Jensen KB, Mulder KW, Watt FM. Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis. Development. 2009; 136: 2815-23. https://doi.org/10.1242/dev.038620.

[22] Zimoch J, Zielinska D, Michalak-Micka K, Rütsche D, Böni R, Biedermann T, et al. Bio-engineering a prevascularized human trilayered skin substitute containing a hypodermis. Acta Biomater. 2021; 134: 215-27. https://doi.org/10.1016/j.actbio.2021.07.033.

[23] Glover JD, Wells KL, Matthäus F, Painter KJ, Ho W, Riddell J, et al. Hierarchical patterning modes orchestrate hair follicle morphogenesis. PLoS Biol. 2017; 15: e2002117. https://doi.org/10.1371/journal.pbio.2002117.

[24] Chen R, Miao Y, Hu Z. Dynamic nestin expression during hair follicle maturation and the normal hair cycle. Mol Med Rep. 2019; 19: 549-54. https://doi.org/10.3892/mmr.2018.9691.

[25] Schmidt BA, Horsley V. Intradermal adipocytes mediate fibroblast recruitment during skin wound healing. Development. 2013; 140: 1517-27. https://doi.org/10.1242/dev.087593.

[26] Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature. 2015; 526: 564-8. https://doi.org/10.1038/nature15695.

[27] Yoshida S, Miwa H, Kawachi T, Kume S, Takahashi K. Generation of intestinal organoids derived from human pluripotent stem cells for drug testing. Sci Rep. 2020; 10: 5989. https://doi.org/10.1038/s41598-020-63151-z.

[28] Abo KM, de Aja JS, Lindstrom-Vautrin J, Alysandratos K-D, Richards A, Garcia-de-Alba C, et al. Air-liquid interface culture promotes maturation and allows environmental exposure of pluripotent stem cell-derived alveolar epithelium. JCI Insight. 2022; 7. https://doi.org/10.1172/jci.insight.155589.

[29] Rosdy M, Clauss L-C. Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface. J Invest Dermatol. 1990; 95: 409-14. https://doi.org/10.1111/1523-1747.ep12555510.

[30] Madaan A, Verma R, Singh AT, Jaggi M. Review of hair follicle dermal papilla cells as in vitro screening model for hair growth. Int J Cosmet Sci. 2018; 40: 429-50. https://doi.org/10.1111/ics.12489.

[31] Yang Z, Liu J, Zhu N, Qi F. Comparison between hair follicles and split-thickness skin grafts in cutaneous wound repair. Int J Clin Exp Med. 2015; 8: 15822-7.

[32] Plotczyk M, Jiménez F, Limbu S, Boyle CJ, Ovia J, Almquist BD, et al. Anagen hair follicles transplanted into mature human scars remodel fibrotic tissue. NPJ Regenerative Medicine. 2023; 8: 1. https://doi.org/10.1038/s41536-022-00270-3.

[33] Plotczyk M, Jimenez F. Hair Follicles in Wound Healing and Skin Remodelling, Vol. 10. Hair Follicle Regeneration: Springer, 2022. 153-63 https://doi.org/10.1089/wound.2019.1139.

[34] Stabell AR, Lee GE, Jia Y, Wong KN, Wang S, Ling J, et al. Single-cell transcriptomics of human-skin-equivalent organoids. Cell Rep. 2023; 42: 112511. https://doi.org/10.1016/j.celrep.2023.112511.