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Volume 42 Issue 6
Jun.  2026
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Article Navigation >  CHINESE JOURNAL OF BURNS AND WOUNDS  > 2026  >  42(6): 542-551
Xia JZ,Wang ZX,Sun D,et al.Culture and identification of human induced pluripotent stem cell-derived skin organoids[J].Chin J Burns Wounds,2026,42(6):542-551.DOI: 10.3760/cma.j.cn501225-20260205-00074.
Citation: Xia JZ,Wang ZX,Sun D,et al.Culture and identification of human induced pluripotent stem cell-derived skin organoids[J].Chin J Burns Wounds,2026,42(6):542-551.DOI: 10.3760/cma.j.cn501225-20260205-00074.
shu

Culture and identification of human induced pluripotent stem cell-derived skin organoids

doi: 10.3760/cma.j.cn501225-20260205-00074
  • 1.

    Department of Plastic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China

  • 2.

    Puncture (Shanghai) Intelligent Medical Technology Co., Ltd., Shanghai 201601, China

Funds:

National Key Research and Development Program of China 2024YFC3407600

International (Regional) Cooperation and Exchange Program of the National Natural Science Foundation of China 82020108020

Youth Fund of the National Natural Science Foundation of China 82322046, 82503043

General Program of National Natural Science Foundation of China 82072198

Open Foundation of Hubei Key Laboratory of Regenerative Medicine and Multi-disciplinary Translational Research 2026AFB714, 2024zsyx08, 2024KQHM02

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    Abstract
  •   Objective  To establish and identify a culture system for skin organoids based on human induced pluripotent stem cells (hiPSCs).  Methods  This study was a basic research with a self-controlled pre-post design. Human primary skin fibroblasts (Fbs) were obtained from discarded hair follicle tissue of a 45-year-old male patient undergoing hair transplant surgery at Tongji Medical College of Huazhong University of Science and Technology. Primary skin Fbs were induced into hiPSCs, which were then cultured to form three-dimensional aggregates. Then the induced differentiation was performed as followings. On differentiation day 0 (the day of differentiation initiation), the three-dimensional aggregates were transferred to ultra-low attachment culture plate and cultured in E6 medium supplemented with Matrigel, transforming growth factor-β type Ⅰ receptor inhibitor of SB431542, basic fibroblast growth factor (bFGF), and bone morphogenetic protein-4 to induce non-neural ectoderm formation. On differentiation day 3, one-quarter volume of E6 medium supplemented with bone morphogenetic protein signaling pathway inhibitor of LDN193189 and bFGF was added to the original medium to continue culturing and to induce the formation of cranial neural crest cells. On differentiation day 6, culture continued with approximately 3/5 volume of plain E6 medium added to the original medium. Half-medium changes were performed on differentiation days 8 and 10 (once each), followed by continued culture. On differentiation day 12, cell clumps were seeded into new ultra-low attachment culture plate to continue culturing, and epidermal self-assembly was induced using organoid maturation medium supplemented with Matrigel. On differentiation day 15, a half-medium change was performed, and culture was continued. On differentiation day 18, the original medium was replaced with organoid maturation medium supplemented with α-melanocyte-stimulating hormone to continue culturing. Starting from differentiation day 21, half-medium changes were performed every 3 days, and culture continued. After differentiation initiation, the stage-specific morphological characteristics of hiPSC differentiation into skin organoids were observed daily. Immunofluorescence staining was performed to assess skin organoids on different days of differentiation: on day 12 for the presence of mesenchymal cells, on day 20 for epidermal terminal differentiation, on day 35 for mesenchymal and epithelial structure formation as well as hair follicle stem cell and basal layer keratinocyte related characteristics, on day 55 for dermal papilla formation, on day 75 for hair germ-like structure formation, and on day 90 for organoid proliferative activity. Fluorescence probe staining was performed on differentiation day 110 to detect hair follicle formation, as well as lipid deposition and sebaceous gland-like structure formation.  Results  On differentiation day 0, hiPSCs formed three-dimensional aggregates with clear boundaries and relatively uniform size. On differentiation day 3, ectoderm-like structures appeared on the surface of the aggregates, accompanied by non-epithelial-like cells beginning to migrate outward. During differentiation days 6 to 8, mesenchymal cells and neuroglial-like cells gradually increased. During differentiation days 12 to 18, organoids with spatial heterogeneity gradually formed within the aggregates, exhibiting preliminary epidermal-dermal-like bilayer structures. On differentiation day 60, hair germ-like structures were observed. During differentiation days 80 to 130, more mature hair germ-like structures and hair follicle-like structures gradually emerged. Immunofluorescence detection showed that on differentiation day 12, early mesenchymal cells were observed around the skin organoids. On differentiation day 20, keratinocytes emerged in the outer layer of the skin organoids. On differentiation day 35, dermal-like cells emerged in the skin organoids, forming distinct spatial compartments with epithelial-like structures, accompanied by the presence of basal layer-like cell populations. On differentiation day 55, dermal papilla-like cell populations were detected and were spatially adjacent to epidermal regions. On differentiation day 75, thickened epithelial-like structures growing inward were observed in localized areas of the skin organoids, along with cell aggregation forming hair germ-like structures; dermal papilla-like cell populations were detected in regions adjacent to the hair germ and were spatially adjacent to epithelial structures. On differentiation day 90, proliferating cell enrichment was observed in the hair germ region. Fluorescence probe staining showed that by differentiation day 110, more mature hair follicle-like structures, hair shaft-like protrusions, lipid deposition, and sebaceous gland-like structures were observed in the skin organoids.  Conclusions  By stage-wise modulation of key signaling pathways, we successfully established a hiPSC-based a culture system for skin organoids. The induced differentiation process of this system highly mimics the in vivo developmental program of skin and hair follicles, resulting in the reconstitution of a complex in vitro skin model with hair follicle-like structures.

     

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    • References(40)
  • [1]
    ProkschE, BrandnerJM, JensenJM. The skin: an indispensable barrier[J]. Exp Dermatol, 2008,17(12):1063-1072. DOI: 10.1111/j.1600-0625.2008.00786.x.
    [2]
    罗高兴, 周璇. 先进生物材料在创面修复中的应用[J].中华烧伤与创面修复杂志,2024,40(1):26-32. DOI: 10.3760/cma.j.cn501225-20231128-00211.
    [3]
    JiS, ZhuZ, SunX, et al. Functional hair follicle regeneration: an updated review[J]. Signal Transduct Target Ther, 2021,6(1):66. DOI: 10.1038/s41392-020-00441-y.
    [4]
    赵思远, 李伟, 孔维诗, 等. 皮肤类器官在创面修复中的应用研究进展[J].中华烧伤与创面修复杂志,2025,41(7):703-707. DOI: 10.3760/cma.j.cn501225-20240901-00323.
    [5]
    刘志欣, 仇恺真, 何佳, 等. 人诱导多能干细胞来源的皮肤类器官条件培养基对高糖诱导的人真皮成纤维细胞功能的影响[J].中华烧伤与创面修复杂志,2025,41(3):286-294. DOI: 10.3760/cma.j.cn501225-20241020-00404.
    [6]
    LeeJ, van der ValkWH, SerdySA, et al. Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells[J]. Nat Protoc, 2022,17(5):1266-1305. DOI: 10.1038/s41596-022-00681-y.
    [7]
    MadisonKC. Barrier function of the skin: "la raison d'être" of the epidermis[J]. J Invest Dermatol, 2003,121(2):231-241. DOI: 10.1046/j.1523-1747.2003.12359.x.
    [8]
    JoostS, AnnusverK, JacobT, et al. The molecular anatomy of mouse skin during hair growth and rest[J]. Cell Stem Cell, 2020,26(3):441-457.e7. DOI: 10.1016/j.stem.2020.01.012.
    [9]
    FuchsE. Scratching the surface of skin development[J]. Nature, 2007,445(7130):834-842. DOI: 10.1038/nature05659.
    [10]
    胡大海, 李梦洋, 王鹏. 复杂创面修复前沿进展:从微环境调控到精准医疗实践[J].中华烧伤与创面修复杂志,2025,41(5):417-425. DOI: 10.3760/cma.j.cn501225-20250407-00171.
    [11]
    WangW, LiuP, ZhuW, et al. Skin organoid transplantation promotes tissue repair with scarless in frostbite[J]. Protein Cell, 2025,16(4):240-259. DOI: 10.1093/procel/pwae055.
    [12]
    TottoliEM, DoratiR, GentaI, et al. Skin wound healing process and new emerging technologies for skin wound care and regeneration[J]. Pharmaceutics, 2020,12(8):735. DOI: 10.3390/pharmaceutics12080735.
    [13]
    SchneiderMR, Schmidt-UllrichR, PausR. The hair follicle as a dynamic miniorgan[J]. Curr Biol, 2009,19(3):R132-142. DOI: 10.1016/j.cub.2008.12.005.
    [14]
    KageyamaT, ShimizuA, AnakamaR, et al. Reprogramming of three-dimensional microenvironments for in vitro hair follicle induction[J]. Sci Adv, 2022,8(42):eadd4603. DOI: 10.1126/sciadv.add4603.
    [15]
    SunJ, AhmedI, BrownJ, et al. The empowering influence of air-liquid interface culture on skin organoid hair follicle development[J/OL]. Burns Trauma, 2025,13:tkae070[2026-02-05]. https://pubmed.ncbi.nlm.nih.gov/39822647/. DOI: 10.1093/burnst/tkae070.
    [16]
    LeeJ, BӧsckeR, TangPC, et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids[J]. Cell Rep, 2018,22(1):242-254. DOI: 10.1016/j.celrep.2017.12.007.
    [17]
    石傲, 王运帷, 康宇晨, 等. 水凝胶促进创面血管化的研究进展[J].中华烧伤与创面修复杂志,2025,41(3):295-300. DOI: 10.3760/cma.j.cn501225-20240521-00193.
    [18]
    LeeJ, RabbaniCC, GaoH, et al. Hair-bearing human skin generated entirely from pluripotent stem cells[J]. Nature, 2020,582(7812):399-404. DOI: 10.1038/s41586-020-2352-3.
    [19]
    TakahashiK, TanabeK, OhnukiM, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J]. Cell, 2007,131(5):861-872. DOI: 10.1016/j.cell.2007.11.019.
    [20]
    EstebanMA, WangT, QinB, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells[J]. Cell Stem Cell, 2010,6(1):71-79. DOI: 10.1016/j.stem.2009.12.001.
    [21]
    StanariusA, Faber-ZuschratterH, TöpelI, et al. Tyramide signal amplification in brain immunocytochemistry: adaptation to electron microscopy[J]. J Neurosci Methods, 1999,88(1):55-61. DOI: 10.1016/s0165-0270(99)00012-6.
    [22]
    LiW, GermainRN, GernerMY. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging[J]. Nat Protoc, 2019,14(6):1708-1733. DOI: 10.1038/s41596-019-0156-4.
    [23]
    ItohM, Umegaki-AraoN, GuoZ, et al. Generation of 3D skin equivalents fully reconstituted from human induced pluripotent stem cells (iPSCs)[J]. PLoS One, 2013,8(10):e77673. DOI: 10.1371/journal.pone.0077673.
    [24]
    DriskellRR, WattFM. Understanding fibroblast heterogeneity in the skin[J]. Trends Cell Biol, 2015,25(2):92-99. DOI: 10.1016/j.tcb.2014.10.001.
    [25]
    SivamaniP, RajendranRL, GangadaranP, et al. An induced pluripotent stem cell-based approach for hair follicle development and regeneration[J]. Regen Ther, 2024,26:502-507. DOI: 10.1016/j.reth.2024.07.005.
    [26]
    中华医学会烧伤外科学分会, 《中华烧伤与创面修复杂志》编辑委员会. 慢性创面外用生长因子的临床专家共识(2025版)[J].中华烧伤与创面修复杂志,2025,41(8):711-724. DOI: 10.3760/cma.j.cn501225-20250426-00191.
    [27]
    KimY, ParkN, RimYA, et al. Establishment of a complex skin structure via layered co-culture of keratinocytes and fibroblasts derived from induced pluripotent stem cells[J]. Stem Cell Res Ther, 2018,9(1):217. DOI: 10.1186/s13287-018-0958-2.
    [28]
    ShafieeA, AtalaA. Tissue engineering: toward a new era of medicine[J]. Annu Rev Med, 2017,68:29-40. DOI: 10.1146/annurev-med-102715-092331.
    [29]
    QuílezC, JeonEY, PappalardoA, et al. Efficient generation of skin organoids from pluripotent cells via defined extracellular matrix cues and morphogen gradients in a spindle-shaped microfluidic device[J]. Adv Healthc Mater, 2024,13(20):e2400405. DOI: 10.1002/adhm.202400405.
    [30]
    WangXY, JiaQN, LiJ, et al. Organoids as tools for investigating skin aging: mechanisms, applications, and insights[J]. Biomolecules, 2024,14(11):1436. DOI: 10.3390/biom14111436.
    [31]
    YangR, ZhengY, LiL, et al. Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors[J]. Nat Commun, 2014,5:5807. DOI: 10.1038/ncomms6807.
    [32]
    HigginsCA, ChenJC, CeriseJE, et al. Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth[J]. Proc Natl Acad Sci U S A, 2013,110(49):19679-19688. DOI: 10.1073/pnas.1309970110.
    [33]
    AbaciHE, CoffmanA, DoucetY, et al. Tissue engineering of human hair follicles using a biomimetic developmental approach[J]. Nat Commun, 2018,9(1):5301. DOI: 10.1038/s41467-018-07579-y.
    [34]
    ZhengZ, HuangM, DuY, et al. Advances in skin organoid technology and their applications across biomedical fields: a review[J]. Organoid Res,2025,1(4):025320026. DOI: 10.36922/OR025320026.
    [35]
    MorassoMI, Tomic-CanicM. Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing[J]. Biol Cell, 2005,97(3):173-183. DOI: 10.1042/BC20040098.
    [36]
    DriskellRR, LichtenbergerBM, HosteE, et al. Distinct fibroblast lineages determine dermal architecture in skin development and repair[J]. Nature, 2013,504(7479):277-281. DOI: 10.1038/nature12783.
    [37]
    RognoniE, WattFM. Skin cell heterogeneity in development, wound healing, and cancer[J]. Trends Cell Biol, 2018,28(9):709-722. DOI: 10.1016/j.tcb.2018.05.002.
    [38]
    FeldmanA, MukhaD, MaorII, et al. Blimp1+ cells generate functional mouse sebaceous gland organoids in vitro[J]. Nat Commun, 2019,10(1):2348. DOI: 10.1038/s41467-019-10261-6.
    [39]
    DiaoJ, LiuJ, WangS, et al. Sweat gland organoids contribute to cutaneous wound healing and sweat gland regeneration[J]. Cell Death Dis, 2019,10(3):238. DOI: 10.1038/s41419-019-1485-5.
    [40]
    LiangL, ZhouJ, WangW, et al. Spatially resolved proteomic mapping in skin organoid for hair follicle development[J]. Mol Cell Proteomics, 2026,25(1):101482. DOI: 10.1016/j.mcpro.2025.101482.
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