-
摘要:
目的 建立一种基于人诱导多能干细胞（hiPSC）的三维皮肤类器官构建体系。 方法 该研究为基础实验研究。取华中科技大学同济医学院收治的1名45岁、男性患者植发手术过程中遗弃的毛囊组织,获取人原代皮肤成纤维细胞。将人原代皮肤成纤维细胞诱导为hiPSC,并培养形成三维聚集体。然后进行如下诱导分化过程：分化第0天（启动分化当天）,将三维聚集体转移至低粘附培养板中,在添加基质胶、抑制剂SB431542、碱性成纤维细胞生长因子（bFGF）及骨形态发生蛋白-4的E6培养基中培养,诱导非神经外胚层形成；分化第3天,在原培养基的基础上补充1/4原体积的含抑制剂LDN193189和bFGF的E6培养基,继续培养,诱导颅神经嵴细胞形成；分化第6天,在原培养基的基础上补充约1/3原体积的单纯E6培养基,继续培养；分化第8天和第10天分别进行半量换液后继续培养；分化第12天,将细胞团块接种于低粘附培养板中,加入含基质胶的类器官成熟培养基,诱导表皮自组装；分化第15天,进行半量换液后继续培养；分化第18天,将原培养基更换为添加α-黑色素细胞刺激素的类器官成熟培养基,继续培养；自分化第21天起,每3天半量换液一次,继续培养。分化启动后,每天观察hiPSC向皮肤类器官分化的阶段性形态特征。采用免疫荧光法于分化第12、20、35、55、75、90天分别鉴定皮肤类器官的间充质细胞情况、表皮终末分化情况、间充质及上皮结构形成情况、毛囊干细胞及基底层角质形成细胞相关特征情况、真皮乳头形成情况及毛芽样结构形成情况、类器官增殖活性。采用组织化学法,鉴定分化第110天,皮肤类器官的毛囊形成情况及脂质沉积与皮脂腺样结构形成情况。 结果 分化第0天,hiPSC形成的三维聚集体边界清晰、大小均匀；分化第3天,聚集体表面出现外胚层样结构,伴非上皮样细胞开始外迁；分化第6~8天,聚集体中的间充质细胞及神经胶质样细胞逐渐增多；分化第12~18天,聚集体中形成具有空间异质性的类器官,出现初步的表皮-真皮样双层结构；分化第60天,可见毛芽样结构形成；分化第80~130天,逐渐出现更成熟的毛芽样结构和毛囊样结构。免疫荧光法检测显示,分化第12天,类器官周围可见早期间充质细胞。分化第20天,类器官外层出现角质形成细胞。分化第35天,类器官中出现真皮样细胞,并与上皮结构形成明显空间分区；同时存在基底层样细胞群。分化第55天,检测到真皮乳头样细胞群,其与表皮区域呈空间邻近分布。分化第75天,类器官局部可见向内生长的增厚上皮结构,并伴有细胞聚集形成毛芽样结构；毛芽邻近区域可检测到真皮乳头样细胞群,与上皮结构呈空间邻近分布。分化第90天,毛芽区域可见增殖细胞富集。组织化学法检测显示,至分化第110天,类器官中可见更成熟的毛囊样结构、毛干样突起、脂质沉积及皮脂腺样结构形成。 结论 本研究成功构建了一种基于hiPSC的三维皮肤类器官体系。通过阶段性调控关键信号通路,在体外重建了具有毛囊样结构的复杂皮肤组织模型。该体系的诱导分化过程高度模拟了人体内皮肤及毛囊的发育程序,为研究人皮肤发生机制及其在再生医学中的应用,提供了可靠的体外模型。 Abstract:Objective To establish a three-dimensional skin organoid construction system based on human induced pluripotent stem cells (hiPSCs). Methods The study was a basic experimental study. 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. According to referenced methods, primary skin Fbs were reprogrammed into hiPSCs, which were then cultured to form three-dimensional aggregates. The differentiation process was performed as follows. On differentiation day 0 (the day of differentiation initiation), three-dimensional aggregates were transferred to low-adhesion culture plates and cultured in E6 medium supplemented with Matrigel, the inhibitor SB431542, basic fibroblast growth factor (bFGF), and bone morphogenetic protein-4 (BMP-4) to induce non-neural ectoderm formation. On differentiation day 3, one-quarter volume of E6 medium containing the inhibitor LDN193189 and bFGF was added to the original medium, and culture was continued to induce cranial neural crest cell formation. On differentiation day 6, approximately one-third volume of plain E6 medium was added to the original medium, and culture was continued. Half-medium changes were performed on differentiation days 8 and 10, followed by continued culture. On differentiation day 12, cell clumps were seeded into low-adhesion culture plates, and epidermal self-assembly was induced using organoid maturation medium containing 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 containing α-melanocyte-stimulating hormone (α-MSH), and culture was continued. Starting from differentiation day 21, half-medium changes were performed every 3 days, and culture was continued. After differentiation initiation, the stage-specific morphological characteristics of hiPSC differentiation into skin organoids were observed daily. Immunofluorescence was performed on differentiation days 12, 20, 35, 55, 75, and 90 to identify respectively the presence of mesenchymal cells, epidermal terminal differentiation, mesenchymal and epithelial structure formation, hair follicle stem cell and basal layer keratinocyte-related characteristics, dermal papilla formation and hair germ-like structure formation, and organoid proliferative activity. Histochemical staining was performed on differentiation day 110 to identify hair follicle formation, as well as lipid deposition and sebaceous gland-like structure formation. Results Microscopic observation revealed that on differentiation day 0, hiPSCs formed three-dimensional aggregates with clear boundaries and uniform size. On differentiation day 3, surface ectoderm-like structures appeared, accompanied by outward migration of non-epithelial cells. On differentiation days 6–8, mesenchymal cells and neuroglial-like cells gradually increased. On differentiation days 12–18, organoids with spatial heterogeneity formed, exhibiting preliminary epidermal-dermal bilayer structures. On differentiation day 60, hair germ-like structures were observed. On differentiation days 80–130, 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 organoids. On differentiation day 20, keratinocytes appeared in the outer layer of the organoids. On differentiation day 35, dermal-like cells emerged and formed distinct spatial compartments with epithelial 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, locally ingrowing thickened epithelial structures were observed, 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. Histochemical 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 organoids. Conclusions A three-dimensional skin organoid system based on hiPSCs was successfully established. Through stage-specific regulation of key signaling pathways, a complex skin tissue model with hair follicle-like structures was reconstructed in vitro. The induction and differentiation processes of this system closely recapitulate the in vivo developmental programs of human skin and hair follicles, thereby providing a reliable in vitro model for studying human skin morphogenesis and its applications in regenerative medicine. -
Key words:
- Skin /
- Organoids /
- Hair follicle /
- Induced pluripotent stem cells /
- Tissue engineering /
- Cell differentiation
-
参考文献
(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. -
图 1 人诱导多能干细胞向皮肤类器官分化的阶段性形态特征 倒置显微镜。1A.分化第0天 可见边界清晰的三维聚集体 ×40；1B.分化第3天,可见聚集体表面形成外胚层样结构,并伴随周围非上皮样细胞外迁 ×40；1C.分化第8天,可见间充质细胞及神经胶质样细胞占比较大 ×40；1D.分化第18天,可见具有明显空间异质性的类器官,上皮样区域与富含间充质细胞区域呈分区分布 ×40；1E.分化第60天,可见毛芽样结构形成 ×100；1F.分化第130天,可见更成熟的毛囊样结构 ×100
图 2 人诱导多能干细胞诱导分化为表皮-真皮乳头样组织结构的鉴定。2A.分化第12天,类器官外围出现CD34阳性（绿色）的早期间充质细胞 Alexa Fluor 488-4′,6-二脒基-2-苯基吲哚 ×40；2B. 分化第20天,外层出现兜甲蛋白阳性（绿色）的终末分化角质形成细胞 Alexa Fluor 488-4′,6-二脒基-2-苯基吲哚 ×40；2C.分化第35天,可见δ样同源蛋白1阳性（绿色）与Ⅲ型胶原蛋白α1阳性（红色）的真皮样细胞,并与E-钙黏蛋白阳性（白色）的表皮结构形成空间分区 Alexa Fluor 488-Alexa Fluor 594-Alexa Fluor 647-4′,6-二脒基-2-苯基吲哚 ×100；2D.分化第35天,可见细胞角蛋白15阳性（绿色）和细胞角蛋白5阳性（红色）共表达的表皮基底层样细胞 Alexa Fluor 488-Alexa Fluor 594-4′,6-二脒基-2-苯基吲哚 ×100；2E.分化第55天,类器官内SRY盒转录因子2阳性（红色）的真皮乳头样细胞群与细胞角蛋白5阳性（绿色）的表皮区域分布 Alexa Fluor 488- Alexa Fluor 594偶联酪胺荧光染料-4′,6-二脒基-2-苯基吲哚 ×40
图 3 人诱导多能干细胞诱导分化为毛芽、毛囊及脂质沉积情况的鉴定 ×100。3A.分化第75天,类器官表皮局部可见P-钙黏蛋白阳性（红色）聚集,形成毛芽样结构 Alexa Fluor 594-4′,6-二脒基-2-苯基吲哚；3B.分化第75天,SRY盒转录因子2阳性（红色）真皮乳头样细胞群与E-钙黏蛋白阳性（绿色）上皮结构呈空间邻近分布,即毛囊微环境建立 Alexa Fluor 488-Alexa Fluor 594-4′,6-二脒基-2-苯基吲哚；3C.分化第90天,可见毛芽区域存在细胞增殖核抗原Ki67阳性（红色）染色的活跃增殖细胞 Alexa Fluor 594-4′,6-二脒基-2-苯基吲哚；3D.分化第110天,可见成熟毛囊样结构及硼二吡咯甲川荧光阳性（绿色）脂质沉积 硼二吡咯甲川-4′,6-二脒基-2-苯基吲哚
-
下载:
图(4)
计量
- 文章访问数: 7
- HTML全文浏览量: 2
- PDF下载量: 2
- 被引次数: 0
