Self-organizing three-dimensional dermal papilla cell spheroids yield therapeutic extracellular vesicles that target hypertrophic scar regression via the miR-26a-5p/CCNE2 axis

Abstract

Background: Hypertrophic scarring remains a critical challenge in regenerative medicine because of the limited efficacy of current antifibrotic therapies. Although dermal papilla cells (DPCs) exhibit intrinsic scar-inhibitory potential, their therapeutic utility is constrained by rapid replicative senescence and poor scalability in traditional monolayer cultures, necessitating innovative strategies to enhance cellular functionality and manufacturing feasibility. Methods: A self-feeder layer 3D (SFL-3D) platform was established to reprogram primary human DPCs into rejuvenated three-dimensional DPC (tdDPC) spheroids via autocrine–paracrine signalling activation. tdDPC-derived extracellular vesicles (tdDPC-EVs) were isolated from culture supernatants by differential centrifugation. The antifibrotic effects of tdDPC-EVs were systematically evaluated using human scar fibroblasts through scratch wound healing assays, CCK-8 proliferation assays, and fibrotic marker analysis [Western blotting and immunofluorescence staining for α-smooth muscle actin (α-SMA) and collagen I]. Bioinformatics was used to predict key pathways involved in hypertrophic scar (HS) pathogenesis, whereas gain/loss-of-function studies investigated the miR-26a-5p/CCNE2 regulatory axis. Therapeutic validation was performed in a rabbit ear hypertrophic scar model with histopathological and molecular profiling. Results: Compared with conventional 3D cultures, the SFL-3D system demonstrated superior proliferative support, enabling stable tdDPC expansion beyond 10 passages while maintaining high viability and enhanced EV biogenesis. miR-26a-5p-enriched tdDPC-EVs attenuated fibrosis through two mechanisms: (1) silencing CCNE2 to block PI3K/AKT-driven collagen overproduction and (2) suppressing α-SMA+ myofibroblast differentiation. In the rabbit ear HS model, tdDPC-EV administration reduced the scar elevation index and restored the collagen I/III ratio to near-physiological levels. Conclusions: This study positions tdDPC-EVs as a scalable acellular therapy that overcomes the replicative senescence and manufacturing limitations of cellular approaches. The antiscarring efficacy of these EVs, which is mediated by the miR-26a-5p/CCNE2/PI3K/AKT axis, highlights their clinical potential as precision-targeted strategies for hypertrophic scar management. The SFL-3D platform further provides a translatable framework for EV-based regenerative therapeutics.

Keywords

Hypertrophic scar / Extracellular vesicles / Three-dimensional spheroids / Dermal papilla cells / CCNE2 / microRNA-26a-5p

References

[1] Tomtschik J, Anand N, Bustos SS, Martinez-Jorge J, Wyles SP. Practical management of hypertrophic scarring: the mayo clinic experience. Arch Dermatol Res. 2024; 316:77. https://doi.org/10.1007/s00403-023-02802-3.

[2] Bailey J, Schwehr M, Beattie A. Management of keloids and hypertrophic scars. Am Fam Physician. 2024; 110:605-11.

[3] Coentro JQ, Pugliese E, Hanley G, Raghunath M, Zeugolis DI. Current and upcoming therapies to modulate skin scarring and fibrosis. Adv Drug Deliv Rev. 2019; 146:37-59. https://doi.org/10.1016/j.addr.2018.08.009.

[4] Wang YW, Shen K, Sun YL, Cao P, Zhang J, Zhang WF, et al. Extracellular vesicles from 3D cultured dermal papilla cells improve wound healing via Krüppel-like factor 4/vascular endothelial growth factor a-driven angiogenesis. Burns Trauma. 2023; 11:tkad034. https://doi.org/10.1093/burnst/tkad034.

[5] Wang YW, Cai FY, Shi A, Kang YC, Zhao RM, Hu ZH, et al. Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi. 2025; 41:559-68. https://doi.org/10.3760/cma.j.cn501225-20240925-00348.

[6] Wiklander OPB, Brennan MÁ, Lötvall J, Breakefield XO, El Andaloussi S. Advances in therapeutic applications of extracellular vesicles. Sci Transl Med. 2019; 11:eaav8521. https://doi.org/10.1126/scitranslmed.aav8521.

[7] Wang B, Liu XM, Liu ZN, Wang Y, Han X, Lian AB, et al. Human hair follicle-derived mesenchymal stem cells: isolation, expansion, and differentiation. World J Stem Cells. 2020; 12:462-70. https://doi.org/10.4252/wjsc.v12.i6.462.

[8] Ravi M, Paramesh V, Kaviya SR, Anuradha E, Solomon FP. 3D cell culture systems: advantages and applications. J Cell Physiol. 2014; 230:16-26. https://doi.org/10.1002/jcp.24683.

[9] Schmal O, Seifert J, Schäffer TE, Walter CB, Aicher WK, Klein G. Hematopoietic stem and progenitor cell expansion in contact with mesenchymal stromal cells in a hanging drop model uncovers disadvantages of 3D culture. Stem Cells Int. 2016; 2016:4148093.

[10] Jahoda CAB, Whitehouse CJ, Reynolds AJ, Hole N. Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages. Exp Dermatol. 2003; 12:849-59. https://doi.org/10.1111/j.0906-6705.2003.00161.x.

[11] Liao R, Xie B, Cui J, Qi Z, Xue S, Wang Y. E2F transcription factor 1 (E2F1) promotes the transforming growth factor TGF-β1 induced human cardiac fibroblasts differentiation through promoting the transcription of CCNE2 gene. Bioengineered. 2021; 12:6869-77. https://doi.org/10.1080/21655979.2021.1972194.

[12] Mosele F, Stefanovska B, Lusque A, Tran Dien A, Garberis I, Droin N, et al. Outcome and molecular landscape of patients with PIK3CA-mutated metastatic breast cancer. Ann Oncol. 2020; 31:377-86. https://doi.org/10.1016/j.annonc.2019.11.006.

[13] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15:550. https://doi.org/10.1186/s13059-014-0550-8.

[14] Zhang Y, Wang S, Yang Y, Zhao S, You J, Wang J, et al. Scarless wound healing programmed by core-shell microneedles. Nat Commun. 2023; 14:3431. https://doi.org/10.1038/s41467-023-39129-6.

[15] Etich J, Holzer T, Pitzler L, Bluhm B, Brachvogel B . MiR-26a modulates extracellular matrix homeostasis in cartilage. Matrix Biol. 2015; 43:27-34. https://doi.org/10.1016/j.matbio.2015.02.014.

[16] Zhong J, Qin Z, Yu H, Liu X, Li C, Shi J, et al. NPPB prevents postoperative peritoneal adhesion formation by blocking volume-activated Cl− current. Naunyn-Schmiedebergs Arch Pharmacol. 2019; 393:501-10. https://doi.org/10.1007/s00210-019-01740-0.

[17] Zhou Z, Zhao Q, Zhao J, Liu X, Li C, Shi J, et al. Research progress of hair follicle and related stem cells in scar-free wound healing. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2021; 35:241-5. https://doi.org/10.7507/1002-1892.202005086.

[18] Rippa AL, Kalabusheva EP, Vorotelyak EA. Regeneration of dermis: scarring and cells involved. Cells. 2019; 8:607. https://doi.org/10.3390/cells8060607.

[19] Qi SH, Liu P, Xie JL, Shu B, Xu YB, Ke CN, et al. Experimental study on repairing of nude mice skin defects with composite skin consisting of xenogeneic dermis and epidermal stem cells and hair follicle dermal papilla cells. Burns. 2008; 34:385-92. https://doi.org/10.1016/j.burns.2007.04.003.

[20] Leirós GJ, Kusinsky AG, Drago H, Bossi S, Sturla F, Castellanos ML, et al. Dermal papilla cells improve the wound healing process and generate hair bud-like structures in grafted skin substitutes using hair follicle stem cells. Stem Cells Transl Med. 2014; 3:1209-19. https://doi.org/10.5966/sctm.2013-0217.

[21] Yang CC, Cotsarelis G. Review of hair follicle dermal cells. J Dermatol Sci. 2010; 57:2-11. https://doi.org/10.1016/j.jdermsci.2009.11.005.

[22] Ohyama M, Zheng Y, Paus R, Stenn KS. The mesenchymal component of hair follicle neogenesis: background, methods and molecular characterization. Exp Dermatol. 2010; 19:89-99. https://doi.org/10.1111/j.1600-0625.2009.00935.x.

[23] Hu S, Li Z, Lutz H, Huang K, Su T, Cores J, et al. Dermal exosomes containing miR-218-5p promote hair regeneration by regulating β-catenin signaling. Sci Adv. 2020; 6:eaba1685. https://doi.org/10.1126/sciadv.aba1685.

[24] Koledova ZS. 3D cell culture: techniques for and beyond organoid applications. Methods Mol Biol. 2024; 2764:1-12. https://doi.org/10.1007/978-1-0716-3674-9_1.

[25] González EAP. Heterogeneity in adipose stem cells. In: Birbrair A (ed). Stem Cells Heterogeneity-Novel Concepts Advances in Experimental Medicine and Biology. Cham: Springer International Publishing, 2019, 119-50.

[26] Luo L, Zhang W, Wang J, Zhao M, Shen K, Jia Y, et al. A novel 3D culture model of human ASCs reduces cell death in spheroid cores and maintains inner cell proliferation compared with a nonadherent 3D culture. Front Cell Dev Biol. 2021; 9:737275. https://doi.org/10.3389/fcell.2021.737275.

[27] Luo L, Zhang W, Chen W, Fu X, Wang X, Xu R, et al. Based on a self-feeder layer, a novel 3D culture model of human ADSCs facilitates trans-differentiation of the spheroid cells into neural progenitor-like cells using siEID3 with a laminin/poly-d-lysine matrix. Cells. 2021; 10:493. https://doi.org/10.3390/cells10030493.

[28] Yin S, Zhou S, Ren D, Zhang J, Xin H, He X, et al. Mesenchymal stem cell-derived exosomes attenuate epithelial-mesenchymal transition of HK-2 cells. Tissue Eng Part A. 2022; 28:651-9. https://doi.org/10.1089/ten.tea.2021.0190.

[29] Kamolz LP, Hecker A. Molecular mechanisms related to burns, burn wound healing and scarring. Int J Mol Sci. 2023; 24:8785. https://doi.org/10.3390/ijms24108785.

[30] Mony MP, Harmon KA, Hess R, Dorafshar AH, Shafikhani SH. An updated review of hypertrophic scarring. Cells. 2023; 12:678. https://doi.org/10.3390/cells12050678.

[31] Angelini A, Trial J, Ortiz-Urbina J, Cieslik KA. Mechanosensing dysregulation in the fibroblast: a hallmark of the aging heart. Ageing Res Rev. 2020; 63:101150. https://doi.org/10.1016/j.arr.2020.101150.

[32] Hinz B. Myofibroblasts. Exp Eye Res. 2016; 142:56-70. https://doi.org/10.1016/j.exer.2015.07.009.

[33] Wang J, Zhao M, Zhang H, Yang F, Luo L, Shen K, et al. KLF4 alleviates hypertrophic scar fibrosis by directly activating BMP4 transcription. Int J Biol Sci. 2022; 18:3324-36. https://doi.org/10.7150/ijbs.71167.

[34] Zhao W, Zhang R, Zang C, Zhang L, Zhao R, Li Q, et al. Exosome derived from mesenchymal stem cells alleviates pathological scars by inhibiting the proliferation, migration and protein expression of fibroblasts via delivering miR-138-5p to target SIRT1. Int J Nanomedicine. 2022; 17:4023-38. https://doi.org/10.2147/IJN.S377317.

[35] Liao R, Qi Z, Tang R, Wang R, Wang Y. Methyl ferulic acid attenuates human cardiac fibroblasts differentiation and myocardial fibrosis by suppressing pRB-E2F1/CCNE2 and RhoA/ROCK2 pathway. Front Pharmacol. 2021; 12:714390. https://doi.org/10.3389/fphar.2021.714390.

[36] Wang S, Lv T, Chen Q, Yang Y, Xu L, Zhang X, et al. Transcriptome sequencing and lncRNA-miRNA-mRNA network construction in cardiac fibrosis and heart failure. Bioengineered. 2022; 13:7118-33. https://doi.org/10.1080/21655979.2022.2045839.

[37] Zhao J, Jiang Q, Xu C, Jia Q, Wang H, Xue W, et al. MiR-26a-5p from HucMSC-derived extracellular vesicles inhibits epithelial mesenchymal transition by targeting Adam17 in silica-induced lung fibrosis. Ecotoxicol Environ Saf. 2023; 257:114950. https://doi.org/10.1016/j.ecoenv.2023.114950.

[38] Li K, Sun H, Bo Y, Zhang W, Huang W, Zhao Y, et al. MCPIP1 promotes atrial remodeling by exacerbating miR-26a-5p/FRAT/Wnt axis-mediated atrial fibrosis in a rat model susceptible to atrial fibrillation. FASEB J. 2024; 38:e70068. https://doi.org/10.1096/fj.202400546RR.

[39] Chung YH, Huang GK, Kang CH, Cheng YT, Kao YH, Chien YS. MicroRNA-26a-5p restoration ameliorates unilateral ureteral obstruction-induced renal fibrosis in mice through modulating TGF-β signaling. Lab Invest. 2023; 103:100131. https://doi.org/10.1016/j.labinv.2023.100131.

[40] Zhang Z, Shang J, Yang Q, Dai Z, Liang Y, Lai C, et al. Exosomes derived from human adipose mesenchymal stem cells ameliorate hepatic fibrosis by inhibiting PI3K/Akt/mTOR pathway and remodeling choline metabolism. J Nanobiotechnol. 2023; 21:29.

[41] He T, Zhang Y, Liu Y, Wang H, Zhang W, Liu J, et al. MicroRNA-494 targets PTEN and suppresses PI3K/AKT pathway to alleviate hypertrophic scar formation. J Mol Histol. 2019; 50:315-23. https://doi.org/10.1007/s10735-019-09828-w.

[42] Wu X, Li J, Yang X, Bai X, Shi J, Gao J, et al. miR-155 inhibits the formation of hypertrophic scar fibroblasts by targeting HIF-1α via PI3K/AKT pathway. J Mol Histol. 2018; 49:377-87. https://doi.org/10.1007/s10735-018-9778-z.

[43] Xiao Y. MiR-486-5p inhibits the hyperproliferation and production of collagen in hypertrophic scar fibroblasts via IGF1/PI3K/AKT pathway. J Dermatolog Trea. 2020; 32:973-82. https://doi.org/10.1080/09546634.2020.1728210.

[44] Zhao S, Liu H, Wang H, He X, Tang J, Qi S, et al. Inhibition of phosphatidylinositol 3-kinase catalytic subunit alpha by miR-203a-3p reduces hypertrophic scar formation via phosphatidylinositol 3-kinase/AKT/mTOR signaling pathway. Burns Trauma. 2024; 12:tkad048. https://doi.org/10.1093/burnst/tkad048.

[45] Wang Q, Wu H, Wu Q, Zhong S. Berberine targets KIF20A and CCNE2 to inhibit the progression of nonsmall cell lung cancer via the PI3K/AKT pathway. Drug Dev Res. 2023; 84:907-21. https://doi.org/10.1002/ddr.22061.

[46] Wu R, Gao Y, Wu J, Wang C, Yang L. Semi-synthetic product dihydroartemisinin inhibited fibronectin-1 and integrin-β1 and interfered with the migration of HCCLM6 cells via PI3K-AKT pathway. Biotechnol Lett. 2020; 42:917-26. https://doi.org/10.1007/s10529-020-02839-8.

[47] Meng X, Yu Z, Xu W, Chai J, Fang S, Min P, et al. Control of fibrosis and hypertrophic scar formation via glycolysis regulation with IR780. Burns Trauma. 2022; 10:tkac015. https://doi.org/10.1093/burnst/tkac015.

[48] Wang M, Qian L, Li J, Ming H, Fang L, Li Y, et al. GHSR deficiency exacerbates cardiac fibrosis: role in macrophage inflammasome activation and myofibroblast differentiation. Cardiovasc Res. 2019; 116:2091-102. https://doi.org/10.1093/cvr/cvz318.