Development of human cartilage circadian rhythm in a stem cell-chondrogenesis model.
Development of human cartilage circadian rhythm in a stem cell-chondrogenesis model.
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DOI：
10.7150/thno.70893
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发表时间：
2022
期刊：
影响因子：
12.4
通讯作者：
Meng, Qing-Jun
中科院分区：
文献类型：
--
作者：
Naven, Mark A.;Zeef, Leo A. H.;Li, Shiyang;Humphreys, Paul A.;Smith, Christopher A.;Pathiranage, Dharshika;Cain, Stuart;Woods, Steven;Bates, Nicola;Au, Manting;Wen, Chunyi;Kimber, Susan J.;Meng, Qing-Jun
小鼠关节软骨中的昼夜节律钟是组织稳态和骨关节炎的关键时间调节机制。然而，这些发现转化为人类已受到阻碍，在获得昼夜节律的时间序列的人软骨组织的困难。因此，需要一个合适的模型来了解人体软骨中昼夜节律的启动和调节。方法：我们使用人胚胎干细胞（hESC）的软骨分化方案作为早期人类软骨细胞发育的代表。使用组织学和多能性和分化标志物的表达来验证软骨发生。通过慢病毒转导人生物钟基因荧光素酶报告基因，在真实的时间内追踪分子生物钟。通过RNAseq和差异表达分析评估分化偶联基因表达。结果：hESC缺乏时钟基因表达的功能性昼夜节律。在软骨形成分化期间，多能性标志物（例如，NANOG和OCT 4）和软骨形成基因（SOX 9、COL2A1和ACAN）的显著增加。在第21天，3D软骨颗粒的组织学显示类似于人软骨的基质结构，具有容易检测的核心时钟蛋白（BMAL1、CLOCK和PER 2）。重要的是，在软骨形成分化方案中，分化hESC的生物钟在第11天（2D阶段结束）和第21天（3D分化后10天）之间被激活。RNA测序揭示了大多数时钟基因和一系列时钟调节因子表达水平的显著分化偶联变化。结论：在人软骨形成模型中，生物钟通过分化偶联机制逐渐激活。这些发现提供了一个人类3D软骨形成模型，以研究生物钟在正常稳态和骨关节炎等疾病中的作用。
The circadian clock in murine articular cartilage is a critical temporal regulatory mechanism for tissue homeostasis and osteoarthritis. However, translation of these findings into humans has been hampered by the difficulty in obtaining circadian time series human cartilage tissues. As such, a suitable model is needed to understand the initiation and regulation of circadian rhythms in human cartilage. Methods: We used a chondrogenic differentiation protocol on human embryonic stem cells (hESCs) as a proxy for early human chondrocyte development. Chondrogenesis was validated using histology and expression of pluripotency and differentiation markers. The molecular circadian clock was tracked in real time by lentiviral transduction of human clock gene luciferase reporters. Differentiation-coupled gene expression was assessed by RNAseq and differential expression analysis. Results: hESCs lacked functional circadian rhythms in clock gene expression. During chondrogenic differentiation, there was an expected reduction of pluripotency markers (e.g., NANOG and OCT4) and a significant increase of chondrogenic genes (SOX9, COL2A1 and ACAN). Histology of the 3D cartilage pellets at day 21 showed a matrix architecture resembling human cartilage, with readily detectable core clock proteins (BMAL1, CLOCK and PER2). Importantly, the circadian clocks in differentiating hESCs were activated between day 11 (end of the 2D stage) and day 21 (10 days after 3D differentiation) in the chondrogenic differentiation protocol. RNA sequencing revealed striking differentiation coupled changes in the expression levels of most clock genes and a range of clock regulators. Conclusions: The circadian clock is gradually activated through a differentiation-coupled mechanism in a human chondrogenesis model. These findings provide a human 3D chondrogenic model to investigate the role of the circadian clock during normal homeostasis and in diseases such as osteoarthritis.
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影响因子：
3.7
作者：
LeSauter J;Lambert CM;Robotham MR;Model Z;Silver R;Weaver DR
通讯作者：
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Meng, Qing-Jun
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影响因子：
10.5
作者：
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通讯作者：
de Crombrugghe, B