Introduction

Human mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. Previous studies have reported that a single addition of 5-azacytidine (5-AzaC) causes the differentiation of hMSCs towards a myocardial lineage. The aim of this work was to evaluate the effect of 5-AzaC addition frequency on hMSCs priming (i.e., indicating an early genetic differentiation) using two culture environments.

Methods

hMSCs were supplemented with 5-AzaC while cultured in well plates and in microfluidic chips. The impact of 5-AzaC concentration (10 and 20 μM) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPARγ (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated.

Results

Daily delivering 5-AzaC caused a higher upregulation of PPARγ, SOX9 and RUNX2 in comparison to a single dose delivery, both under static well plates and dynamic microfluidic cultures. A particularly high gene expression of PPARγ (tenfold-change) could indicate priming of hMSCs towards adipocytes.

Conclusions

Both macro- and microscale cultures provided results with similar trends, where addition frequency of 5-AzaC was a crucial factor to upregulate several genes. Microfluidics technology was proven to be a suitable platform for the continuous delivery of a drug and could be used for screening purposes in tissue engineering research.

中文翻译：

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5-氮杂胞苷添加频率在微量和大规模培养物中的影响

介绍

人间充质干细胞 (hMSCs) 由于其多向分化能力，因此在组织再生方面具有巨大的临床潜力。以前的研究报道，单次添加 5-氮杂胞苷 (5-AzaC) 会​​导致 hMSCs 向心肌谱系分化。这项工作的目的是使用两种培养环境评估 5-AzaC 添加频率对 hMSCs 启动（即表明早期遗传分化）的影响。

方法

hMSCs 在孔板和微流控芯片中培养时补充了 5-AzaC。5-AzaC 浓度（10 和 20  μM）和添加频率（一次、每天或连续）以及培养期（2 或 5 天）对 PPARγ（脂肪细胞）、PAX3（成肌细胞）遗传上调的影响)、SOX9（软骨细胞）和 RUNX2（成骨细胞）进行了评估。

结果

与单剂量递送相比，在静态孔板和动态微流体培养下，每日递送 5-AzaC 导致 PPARγ、SOX9 和 RUNX2 的上调更高。PPARγ 的特别高基因表达（十倍变化）可能表明 hMSCs 对脂肪细胞的启动。

结论

宏观和微观培养都提供了具有相似趋势的结果，其中 5-AzaC 的添加频率是上调几个基因的关键因素。微流控技术已被证明是一种适用于持续输送药物的平台，可用于组织工程研究中的筛选目的。