Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate./nMethods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions./nResults: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting./nConclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.

中文翻译：

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分化祖细胞的自组装促进球状人体皮肤类器官形成和平面皮肤再生


单细胞自组装实体器官将极大地扩展再生医学的适用性。干/祖细胞可以自组织成微型器官单元，称为类器官，部分模拟组织功能和再生。在这里，我们展示了成体和诱导多能干细胞 (iPSC) 衍生的成纤维细胞、角质形成细胞和内皮祖细胞的 3D 自组装，体内为平面人类皮肤，体外为新型球形皮肤类器官。人血小板裂解物。/n方法：从人体组织中分离原代内皮集落形成细胞 (ECFC)、皮肤成纤维细胞 (FB) 和角质形成细胞 (KC)，并在 2D 无异源条件下进行多克隆增殖。根据效率优化方案，人组织来源的 iPSC 分化为内皮细胞 (hiPSC-EC)、成纤维细胞 (hiPSC-FB) 和角质形成细胞 (hiPSC-KC)。通过流式细胞术证实了细胞身份和纯度，克隆形成性表明了它们的干/祖细胞潜力。含有人血小板衍生生长因子的培养条件促进了三细胞型漂浮球体的形成，并使用纳米颗粒细胞标记来监测组织过程。在移植 hiPSC 衍生的单细胞悬浮液后，对免疫缺陷小鼠的全层伤口的平面人类皮肤再生进行了评估。/n 结果：类器官显示出独特的结构，表面锚定的角质形成细胞围绕基质核心，并且在基质中具有特定的信号传导模式。对炎症刺激的反应。 FGF-7 mRNA 转染需要加速角质形成细胞的长期健康。 在成人或 iPSC 衍生的皮肤细胞悬浮液体移植后两周内，分层的人类皮肤也会自组装，从而治愈小鼠的深层伤口。在共移植内皮祖细胞的情况下，移植血管化显着加速。从机制上讲，细胞外囊泡介导多因素血小板衍生的营养作用。异种移植后没有发生肿瘤发生。/n结论：这说明了高级祖细胞自组织原理，并允许利用浮动球体皮肤类器官进行新型快速 3D 皮肤相关药物高内涵测试。多细胞移植自组织利用人血小板因子支持的细胞悬浮移植促进基于 iPSC 的器官再生策略的开发。