Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates
Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates
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DOI：
10.1007/s00441-016-2405-y
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发表时间：
2016-04
影响因子：
3.6
通讯作者：
Chizuka Obara;Ken-ichi Tomiyama;Kazuya Takizawa;R. Islam;Takeshi Yasuda;Takaya Gotoh;Katsushi Tajima-Katsushi-Tajim
中科院分区：
文献类型：
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作者：
Chizuka Obara;Ken-ichi Tomiyama;Kazuya Takizawa;R. Islam;Takeshi Yasuda;Takaya Gotoh;Katsushi Tajima-Katsushi-Tajim
三维聚集体培养对于研究间充质干细胞/基质细胞（MSCs）的功能特性是有用的。然而，对于3-D间充质干细胞分析，必须首先进行二维（2-D）单层培养的间充质干细胞预扩增，这可能会取消其内源性特性。为了避免对二维扩增的需要，我们使用了前瞻性分离的小鼠骨髓(BM)-MSC，并检查了二维和三维MSC培养物的生物学特性差异。BM-MSCs在纳米培养板（NCP）上自组装成聚集体，纳米培养板具有低细胞结合纹理的纳米印迹图案。仅通过扩散培养，3-D MSCs的增殖速度与2- d培养细胞相同，并且分泌更高水平的促血管生成因子，如血管内皮生长因子和肝细胞生长因子（HGF）。在试管形成实验中，3-D间充质干细胞培养的条件培养基比2-D间充质干细胞促进更多的毛细血管形成。基质植入的3d间充质干细胞聚集体倾向于诱导宿主小鼠血管生成。NCP的三维培养诱导了MSCs中甲胎蛋白（AFP）的表达，而不使用甲胎蛋白或内胚层诱导因子，可能是通过hgf自分泌机制，并保持了它们向脂肪细胞、骨细胞和软骨细胞分化的能力。前瞻性分离的小鼠BM-MSCs表达低/负的干细胞相关基因，包括Oct3/4、Nanog和Sox2，这些基因在基于ncp的三维培养中没有增强，这表明其中一些细胞分化为中内胚层细胞。在NCP上3d培养预期分离的MSCs可以分析更内源性的BM-MSCs的生物学特性，并可能有助于组织工程和修复。
Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair.