Department of Industrial Engineering (DII), University of Padua, via Marzolo 9, 35131 Padua, Italy.
Biotechnol Bioeng. 2012 Dec;109(12):3119-32. doi: 10.1002/bit.24571. Epub 2012 Jun 20.
The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self-organization into germ layers. In this work, we aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification.
人多能干细胞（hPS）的治疗潜力受到各种问题的威胁，其中包括难以将细胞分化均匀地定向为特定谱系。从 hPSC 向特化的分化细胞的转变伴随着分泌组活性、细胞外基质的重塑以及向胚层的自我组织。在这项工作中，我们旨在研究不同的三维微环境如何调节源自人胚胎干细胞（hES）的胚状体中的三个胚层的早期分化。特别是，专门设计了一种可渗透、生物相容的水凝胶微井阵列，以重现一个受限的小生境，其中 EB 分泌的分子根据水凝胶的扩散截止而聚集。荧光恢复后光漂白技术用于准确评估水凝胶的渗透性、网格尺寸和可扩散分子的扩散截止值。分析了三种不同的 EB 培养条件：悬浮培养、宽度/深度比为 1:1 和 1:2 的微井中培养。结果表明，在微井中培养的 EB 在培养 8 天后仍然存活且平均大小相当。全基因组微阵列显示，悬浮和受限 EB 培养之间观察到显著的差异基因表达。特别是，在微井中培养的 EB 促进了参与模式特化过程、大脑发育、外胚层和内胚层分化的基因的表达。相反，悬浮培养的 EB 则表达中胚层特化和心脏发育相关的基因。这些结果表明，EB 分泌分子的局部积累驱动了分化模式，这一点通过水凝胶受限 EB 培养中的胚层标记的免疫荧光得到了证实，无论是从 hES 细胞还是人诱导多能干细胞（hiPS）细胞中培养的 EB 都得到了证实。我们的发现强调了生物材料在通过分泌因子小生境特化控制 hPSC 分化方面的额外潜在作用。
