Cell Therapy Center, China Medical University Hospital, Taichung 404332, Taiwan.
Department of Orthopedics, China Medical University Hospital, Taichung 404332, Taiwan.
Int J Mol Sci. 2021 Mar 15;22(6):2978. doi: 10.3390/ijms22062978.
Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor β1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.
间充质干细胞（MS）、胚胎干细胞（ES）和诱导多能干细胞（iPS）以其分化为不同谱系的能力而闻名，包括培养中的软骨细胞。然而，现有的软骨细胞分化方案既耗时又费力。为了改进和简化分化策略，我们探讨了生长因子（转化生长因子β 1（Tgfb1）和集落刺激因子 3（Csf3）之间的相互作用以及培养环境（2D 单层和 3D 纳米纤维支架）对软骨分化的影响。为此，我们检查了细胞形态、增殖率、活力和基因表达谱，并在不同处理方案下对软骨形成培养物中形成的软骨基质进行了特征描述。我们的数据表明，3D 培养比 2D 培养支持更高的增殖率。Tgfb1 促进两种类型培养物中的细胞增殖和活力，而 Csf3 仅在 3D 培养物中显示出积极作用。有趣的是，我们的结果表明 Tgfb1 和 Csf3 的联合处理不会影响细胞增殖和活力。不同处理组中软骨基质的表达表明存在软骨细胞。我们发现，在分化阶段 1 结束时，多能标记物下调，而中胚层标记物上调。然而，仅在 3D 培养物中上调了软骨生成标记物（col2a1 和 aggrecan）的表达。在这里，我们报告了一种有效的、可扩展的、方便的 iPS 细胞软骨分化方案，我们的数据表明 3D 培养环境结合 Tgfb1 和 Csf3 处理可促进软骨分化。
