Differentiation of Induced Pluripotent Stem Cells towards Mesenchymal Stromal Cells is Hampered by Culture in 3D Hydrogels [DNA methylation array]. Differentiation of Induced Pluripotent Stem Cells towards Mesenchymal Stromal Cells is Hampered by Culture in 3D Hydrogels [DNA methylation array]

Directed differentiation of induced pluripotent stem cells (iPSCs) towards specific lineages remains a major challenge in regenerative medicine, while there is a growing perception that this process can be influenced by the three-dimensional environment. In this study, we investigated whether iPSCs can differentiate towards mesenchymal stromal cells (MSCs) when embedded into fibrin hydrogels to enable a one-step differentiation procedure within a scaffold. Differentiation of iPSCs on tissue culture plastic or on top of fibrin hydrogels resulted in a typical MSC-like phenotype. In contrast, iPSCs embedded into fibrin gel gave rise to much smaller cells with heterogeneous growth patterns, absence of fibronectin, faint expression of CD73 and CD105, and reduced differentiation potential towards osteogenic and adipogenic lineage. Transcriptomic analysis demonstrated that characteristic genes for MSCs and extracellular matrix were upregulated on flat substrates, whereas genes of neural development were upregulated in 3D culture. Furthermore, the 3D culture had major effects on DNA methylation profiles, particularly within genes for neuronal and cardiovascular development, while there was no evidence for epigenetic maturation towards MSCs. Taken together, iPSCs could be differentiated towards MSCs on tissue culture plastic or on a flat fibrin hydrogel. In contrast, the differentiation process was heterogeneous and not directed towards MSCs when iPSCs were embedded into the hydrogel. Overall design: Three different iPSC lines (donors 1-3) were differentiated toward MSCs either on fibrin gel or within fibrin gel to compare impact of 2D and 3D culture with a soft substrate on DNA methylation profiles with Illumina Infinium MethylationEPIC BeadChip

诱导多能干细胞（induced pluripotent stem cells, iPSCs）定向分化为特定细胞谱系仍是再生医学领域的重大挑战，而越来越多的研究表明，三维微环境可对该分化过程产生调控作用。本研究旨在探究：当将iPSCs包埋于纤维蛋白水凝胶中以实现在支架内一步完成分化流程时，其能否定向分化为间充质基质细胞（mesenchymal stromal cells, MSCs）。
在组织培养塑料表面或纤维蛋白水凝胶上层培养的iPSCs，可分化为典型的类间充质基质细胞表型。与之相反，包埋于纤维蛋白凝胶内的iPSCs则分化为体积更小的细胞，呈现异质性生长模式，不表达纤连蛋白，CD73与CD105的表达微弱，且向成骨细胞和成脂细胞谱系分化的潜能降低。
转录组学分析显示：在平面基底上培养时，间充质基质细胞特征基因与细胞外基质相关基因均呈上调表达；而在三维培养体系中，神经发育相关基因则被显著激活。此外，三维培养体系对DNA甲基化谱具有显著影响，尤其在神经发育与心血管发育相关基因区域；但未观察到向间充质基质细胞方向的表观遗传成熟迹象。
综上，在组织培养塑料表面或平面纤维蛋白水凝胶上层培养时，iPSCs可定向分化为间充质基质细胞；而当iPSCs被包埋于水凝胶内部时，其分化过程呈现异质性，且未定向朝向间充质基质细胞谱系。
实验整体设计：选取3株不同供体来源的iPSCs细胞系（供体1-3），分别在纤维蛋白凝胶表面及内部定向分化为间充质基质细胞，采用Illumina Infinium MethylationEPIC BeadChip检测DNA甲基化谱，以比较软基底的二维与三维培养体系对甲基化谱的影响。