Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, USA.
Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA.
Cells Tissues Organs. 2022;211(3):294-303. doi: 10.1159/000514580. Epub 2021 May 26.
Directed neural differentiation of embryonic stem cells (ESCs) has been studied extensively to improve the treatment of neurodegenerative disorders. This can be done through stromal-cell derived inducing activity (SDIA), by culturing ESCs directly on top of a layer of feeder stromal cells. However, the stem cells usually become mixed with the feeder cells during the differentiation process, making it difficult to obtain a pure population of the differentiated cells for further use. To address this issue, a non-planar microfluidic device is used here to encapsulate murine ESCs (mESCs) in the 3D liquid core of microcapsules with an alginate hydrogel shell of different sizes for early neural differentiation through SDIA, by culturing mESC-laden microcapsules over a feeder layer of PA6 cells. Furthermore, the alginate hydrogel shell of the microcapsules is modified via oxidation or RGD peptide conjugation to examine the mechanical and chemical effects on neural differentiation of the encapsulated mESC aggregates. A higher expression of Nestin is observed in the aggregates encapsulated in small (∼300 μm) microcapsules and cultured over the PA6 cell feeder layer. Furthermore, the modification of the alginate with RGD facilitates early neurite extension within the microcapsules. This study demonstrates that the presence of the RGD peptide, the SDIA effect of the PA6 cells, and the absence of leukemia inhibition factor from the medium can lead to the early differentiation of mESCs with extensive neurites within the 3D microenvironment of the small microcapsules. This is the first study to investigate the effects of cell adhesion and degradation of the encapsulation materials for directed neural differentiation of mESCs. The simple modifications (i.e., oxidation and RGD incorporation) of the miniaturized 3D environment for improved early neural differentiation of mESCs may potentially enhance further downstream differentiation of the mESCs into more specialized neurons for therapeutic use and drug screening.
胚胎干细胞 (ESC) 的定向神经分化已被广泛研究，以改善神经退行性疾病的治疗效果。这可以通过基质细胞衍生的诱导活性 (SDIA) 来实现，即将 ESC 直接培养在一层饲养基质细胞上。然而，在分化过程中，干细胞通常会与饲养细胞混合，因此很难获得纯的分化细胞群体以供进一步使用。为了解决这个问题，这里使用了一种非平面微流控装置，通过 SDIA 将鼠源 ESC (mESC) 包封在不同大小的藻酸盐水凝胶壳的 3D 液体芯微胶囊中，用于早期神经分化，方法是将载有 mESC 的微胶囊培养在 PA6 细胞的饲养层上。此外，通过氧化或 RGD 肽缀合修饰微胶囊的藻酸盐水凝胶壳，研究机械和化学效应对包封的 mESC 聚集体神经分化的影响。在小（约 300μm）微胶囊中包封并在 PA6 细胞饲养层上培养的聚集体中观察到巢蛋白的表达更高。此外，RGD 的修饰促进了微胶囊内早期神经突的延伸。这项研究表明，RGD 肽的存在、PA6 细胞的 SDIA 效应以及培养基中无白血病抑制因子可导致 mESC 在小微胶囊的 3D 微环境中早期分化出大量神经突。这是第一项研究包封材料的细胞黏附和降解对 mESC 定向神经分化的影响的研究。对小型化 3D 环境的简单修饰（即氧化和 RGD 掺入）可改善 mESC 的早期神经分化，可能有助于进一步将 mESC 分化为更专门的神经元，用于治疗和药物筛选。
