Department of Polymer Science, The University of Akron, Akron, Ohio, United States.
Biomacromolecules. 2013 Sep 9;14(9):3047-54. doi: 10.1021/bm4006112. Epub 2013 Aug 7.
Stem cells have shown lineage-specific differentiation when cultured on substrates possessing signaling groups derived from the native tissue. A distinct determinant in this process is the concentration of the signaling motif. While several groups have been working actively to determine the specific factors, concentrations, and mechanisms governing the differentiation process, many have been turning to combinatorial and gradient approaches in attempts to optimize the multiple chemical and physical parameters needed for the next advance. However, there has not been a direct comparison between the cellular behavior and differentiation of human mesenchymal stem cells cultured in gradient and discrete substrates, which quantitates the effect of differences caused by cell-produced, soluble factors due to design differences between the culture systems. In this study, the differentiation of human mesenchymal stem cells in continuous and discrete polyethylene glycol dimethacrylate (PEGDM) hydrogels containing an RGD concentration gradient from 0 to 14 mM were examined to study the effects of the different culture conditions on stem-cell behavior. Culture condition was found to affect every osteogenic (alkaline phosphatase, Runx 2, type 1 collagen, bone sailoprotein, and calcium content) and adipogenic marker (oil red and peroxisome proliferator-activated receptor gamma) examined regardless of RGD concentration. Only in the continuous gradient culture did RGD concentration affect human mesenchymal stem-cell lineage commitment with low RGD concentrations expressing higher osteogenic differentiation than high RGD concentrations. Conversely, high RGD concentrations expressed higher adipogenic differentiation than low RGD concentrations. Cytoskeletal actin organization was only affected by culture condition at low RGD concentrations, indicating that it played a limited role in the differences in lineage commitment observed. Therefore, the role of discrete versus gradient strategies in high-throughput experimentation needs to be considered when designing experiments as we show that the respective strategies alter cellular outcomes even though base scaffolds have similar material and chemical properties.
当在具有源自天然组织的信号基团的基底上培养时，干细胞表现出谱系特异性分化。在这个过程中一个明显的决定因素是信号基序的浓度。虽然有几个小组一直在积极确定具体的因素、浓度和机制来控制分化过程，但许多小组已经转向组合和梯度方法，试图优化下一次进展所需的多种化学和物理参数。然而，在梯度和离散底物中培养的人骨髓间充质干细胞的细胞行为和分化之间，还没有直接进行比较，这量化了由于培养系统设计差异导致细胞产生的可溶性因子引起的差异的影响。在这项研究中，研究了在含有从 0 到 14mM 的 RGD 浓度梯度的连续和离散聚乙二醇二甲基丙烯酸酯 (PEGDM) 水凝胶中培养的人骨髓间充质干细胞的分化，以研究不同培养条件对干细胞行为的影响。研究发现，培养条件影响每一个成骨（碱性磷酸酶、Runx2、I 型胶原、骨钙素和钙含量）和脂肪生成标志物（油红和过氧化物酶体增殖物激活受体γ），而与 RGD 浓度无关。只有在连续梯度培养中，RGD 浓度才会影响人骨髓间充质干细胞的谱系定向，低 RGD 浓度表达出比高 RGD 浓度更高的成骨分化。相反，高 RGD 浓度表达出比低 RGD 浓度更高的脂肪生成分化。细胞骨架肌动蛋白组织仅在低 RGD 浓度下受培养条件影响，表明其在观察到的谱系定向差异中发挥的作用有限。因此，在设计实验时需要考虑离散与梯度策略在高通量实验中的作用，因为我们表明，即使基本支架具有相似的材料和化学性质，各自的策略也会改变细胞结果。
