Culture and identification of human induced pluripotent stem cell-derived skin organoids
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摘要:
目的 建立一种基于人诱导多能干细胞（hiPSC）的皮肤类器官培养体系,并进行鉴定。 方法 该研究为自身前后对照设计的基础研究。取华中科技大学同济医学院收治的1例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：分化第0天（启动分化当天）,将三维聚集体转移至超低吸附培养板中,在添加基质胶、转化生长因子-β Ⅰ型受体抑制剂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原体积的添加骨形态发生蛋白信号通路抑制剂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3/5原体积的单纯E6培养基,继续培养；分化第8天和第10天各进行1次半量换液后继续培养；分化第12天,将细胞团块接种于新的超低吸附培养板中,加入添加基质胶的类器官成熟培养基继续培养,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1次,继续培养。启动分化后,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天检测皮肤类器官的间充质细胞情况,于分化第20天检测表皮终末分化情况,于分化第35天检测间充质及上皮结构形成情况和毛囊干细胞及基底层角质形成细胞相关特征情况,于分化第55天检测真皮乳头细胞形成情况,于分化第75天检测毛芽样结构形成情况,于分化第90天检测类器官增殖活性。分化第110天,采用荧光探针染色检测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、大小相对均匀；分化第3天,聚集体表面出现外胚层样结构,周围伴有非上皮样细胞开始向外迁移；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,聚集体中逐渐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,皮肤类器官周围可见早期间充质细胞。分化第20天,皮肤类器官外层出现角质形成细胞。分化第35天,皮肤类器官中出现真皮样细胞,并与上皮样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,皮肤类器官局部可见向内生长的增厚上皮样结构,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。荧光探针染色检测显示,分化第110天,皮肤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 通过阶段性调控关键信号通路,本研究成功构建了一种基于hiPSC的皮肤类器官培养体系。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,在体外重建了具有毛囊样结构的复杂皮肤组织模型。 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. -
Key words:
- Skin /
- Organoids /
- Hair follicle /
- Induced pluripotent stem cells /
- Tissue engineering /
- Cell differentiation
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参考文献
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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向外迁移 ×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,类器官周围出现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,类器官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）的表皮区域呈空间邻近分布 Alexa Fluor 488-Alexa Fluor 594-4′,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,类器官局部可见P-钙黏蛋白阳性（红色）细胞聚集,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 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）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
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