A Single-Cell Culture System for Dissecting Microenvironmental Signaling in Development and Disease of Cartilage Tissue.
A Single-Cell Culture System for Dissecting Microenvironmental Signaling in Development and Disease of Cartilage Tissue.
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DOI：
10.3389/fcell.2021.725854
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发表时间：
2021
影响因子：
5.5
通讯作者：
Evseenko D
中科院分区：
文献类型：
--
作者：
Tassey J;Sarkar A;Van Handel B;Lu J;Lee S;Evseenko D
软骨组织由细胞外基质和软骨细胞组成，软骨细胞是一种在成人中具有非常低的细胞更新的细胞类型，提供有限的再生能力。然而，在发育过程中，大量的软骨细胞活跃地增殖并重塑周围的基质。解偶联微环境的影响，决定这些细胞的克隆形成潜力和终末分化之间的平衡是至关重要的新方法的发展软骨再生。不幸的是，大多数现有的方法不适用于在一个单一的细胞分辨率的软骨细胞的功能特性的分析。在这里，我们证明了一种新的3D培养方法提供了一个长期的和允许的体外生态位，选择高度克隆形成，集落形成的软骨细胞，保持软骨特异性基质的生产，从而重演体内生态位。作为概念的证明，Sox 9 IRES-EGFP小鼠软骨细胞的克隆形成性几乎仅在已知富含软骨细胞祖细胞的最高GFP+级分中发现。虽然克隆形成软骨细胞在成人软骨中非常罕见，但我们已经优化了该系统，以支持来自成年猪和人关节软骨细胞的具有复杂带状结构和稳健软骨形成表型的大型单细胞衍生的软骨形成类器官。此外，我们已经证明了这些类器官中的生长轨迹和基质生物合成对促炎环境做出了反应。这种培养方法提供了一个强大的，明确的和可控的系统，可进一步用于询问各种微环境信号对软骨细胞的影响，提供了一个高通量平台，以评估发育和疾病中的遗传和环境因素。
Cartilage tissue is comprised of extracellular matrix and chondrocytes, a cell type with very low cellular turnover in adults, providing limited capacity for regeneration. However, in development a significant number of chondrocytes actively proliferate and remodel the surrounding matrix. Uncoupling the microenvironmental influences that determine the balance between clonogenic potential and terminal differentiation of these cells is essential for the development of novel approaches for cartilage regeneration. Unfortunately, most of the existing methods are not applicable for the analysis of functional properties of chondrocytes at a single cell resolution. Here we demonstrate that a novel 3D culture method provides a long-term and permissive in vitro niche that selects for highly clonogenic, colony-forming chondrocytes which maintain cartilage-specific matrix production, thus recapitulating the in vivo niche. As a proof of concept, clonogenicity of Sox9IRES–EGFP mouse chondrocytes is almost exclusively found in the highest GFP+ fraction known to be enriched for chondrocyte progenitor cells. Although clonogenic chondrocytes are very rare in adult cartilage, we have optimized this system to support large, single cell-derived chondrogenic organoids with complex zonal architecture and robust chondrogenic phenotype from adult pig and human articular chondrocytes. Moreover, we have demonstrated that growth trajectory and matrix biosynthesis in these organoids respond to a pro-inflammatory environment. This culture method offers a robust, defined and controllable system that can be further used to interrogate the effects of various microenvironmental signals on chondrocytes, providing a high throughput platform to assess genetic and environmental factors in development and disease.
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影响因子：
2.2
作者：
Matak D;Brodaczewska KK;Lipiec M;Szymanski Ł;Szczylik C;Czarnecka AM
通讯作者：
Czarnecka AM
影响因子：
10.5
作者：
Akiyama, H;Chaboissier, MC;de Crombrugghe, B
通讯作者：
de Crombrugghe, B
影响因子：
4.5
作者：
Leung VY;Gao B;Leung KK;Melhado IG;Wynn SL;Au TY;Dung NW;Lau JY;Mak AC;Chan D;Cheah KS
通讯作者：
Cheah KS
影响因子：
4.8
作者：
Chan, Chun Ming;Macdonald, Christopher D.;Rowan, Andrew D.
通讯作者：
Rowan, Andrew D.
影响因子：
64.5
作者：
Sacchetti, Benedetto;Funari, Alessia;Bianco, Paolo
通讯作者：
Bianco, Paolo