S100B+A1 CELISA: A Novel Potency Assay and Screening Tool for Redifferentiation Stimuli of Human Articular Chondrocytes

Diaz-Romero Jose; Kuersener Sibylle; Kohl SandroNesic Dobrila

摘要

During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell-based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: (i) demonstrate a correlation between S100B+A1-positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; (ii) develop an S100B+A1 cell-based ELISA, and (iii) prove that S100B+A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high-osmolarity/BMP4 treatment in monolayer) using qRT-PCR, immunocytochemistry, or immunohistochemistry. A cell-based ELISA (CELISA) was developed as a 96-well microplate multiplex assay to measure S100B+A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow-derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B+A1-positive HAC in monolayer and their neochondrogenesis in pellets validates S100B+A1 as a marker of chondrogenic potency. The S100B+A1-CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high-throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets-induced with high-osmolarity and BMP4 treatment in monolayer-suggests that cell instruction prior to implantation may improve cartilage repair. (C) 2016 Wiley Periodicals, Inc.

机译：在单层的扩张,一个必要的一步自体软骨细胞移植,人类关节软骨细胞去分化和(HAC)失去他们的生产能力稳定的透明软骨。如何触发他们的再分化提高细胞软骨再生疗法。S100A1蛋白作为HAC的标记再分化的潜能。(我)证明之间的关联在单层培养和S100B + A1-positive工厂他们在小球neochondrogenesis能力文化;ELISA和(iii)证明S100B + A1感应HAC chondrogenic能力增加。S100A1和S100B的表达模式调查在HAC去分化(单层)或再分化(颗粒或在单层high-osmolarity / BMP4治疗)使用中存在,免疫细胞化学,或免疫组织化学。已经开发成一个微型板块多路96 -好吗分析方法来衡量S100B + A1(软骨形成),碱性磷酸酶(肥大)和DNA(标准化),应用于肝,骨头骨髓来源间充质干细胞和ATDC5 chondrocytic细胞系。的百分比之间的相关性单层和S100B + A1-positive工厂neochondrogenesis在球团矿验证S100B + A1chondrogenic效力的一个标志。S100B + HAC A1-CELISA准确确定分化状态,允许的识别chondrogenic刺激和允许同时监测不良肥厚性表型。高通量、全面和多才多艺方法测量细胞chondrogenic效力和识别再分化因素和条件。在pellets-induced neochondrogenesishigh-osmolarity BMP4治疗monolayer-suggests细胞指令之前移植可以改善软骨修复。2016年威利期刊、公司。

S100B+A1 CELISA: A Novel Potency Assay and Screening Tool for Redifferentiation Stimuli of Human Articular Chondrocytes

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