McKee Christina, Brown Christina, Bakshi Shreeya, Walker Keegan, Govind Chhabi K, Chaudhry G Rasul
Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA.
OU-WB Institute for Stem Cell and Regenerative Medicine, Rochester, MI 48309, USA.
Biomolecules. 2020 Dec 28;11(1):21. doi: 10.3390/biom11010021.
Naïve human embryonic stem cells (ESCs) are characterized by improved viability, proliferation, and differentiation capacity in comparison to traditionally derived primed human ESCs. However, currently used two-dimensional (2-D) cell culture techniques fail to mimic the three-dimensional (3-D) in vivo microenvironment, altering morphological and molecular characteristics of ESCs. Here, we describe the use of 3-D self-assembling scaffolds that support growth and maintenance of the naïve state characteristics of ESC line, Elf1. Scaffolds were formed via a Michael addition reaction upon the combination of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups. 3-D scaffold environment maintained the naïve state and supported the long-term growth of ESCs. RNA-sequencing demonstrated significant changes in gene expression profiles between 2-D and 3-D grown cells. Gene ontology analysis revealed upregulation of biological processes involved in the regulation of transcription and translation, extracellular matrix organization, and chromatin remodeling in 3-D grown cells. 3-D culture conditions also induced upregulation of genes associated with Wnt and focal adhesion signaling, while p53 signaling pathway associated genes were downregulated. Our findings, for the first time, provide insight into the possible mechanisms of self-renewal of naïve ESCs stimulated by the transduction of mechanical signals from the 3-D microenvironment.
与传统来源的始发态人类胚胎干细胞相比，未经处理的人类胚胎干细胞（ESCs）具有更强的活力、增殖能力和分化能力。然而，目前使用的二维（2-D）细胞培养技术无法模拟三维（3-D）体内微环境，从而改变了胚胎干细胞的形态和分子特征。在此，我们描述了使用三维自组装支架来支持胚胎干细胞系Elf1的原始状态特征的生长和维持。支架是通过巯基（PEG-8-SH）和丙烯酸酯（PEG-8-Acr）端基功能化的两种八臂聚乙二醇（PEG）聚合物结合后发生迈克尔加成反应形成的。三维支架环境维持了原始状态并支持胚胎干细胞的长期生长。RNA测序显示二维和三维培养的细胞之间基因表达谱有显著变化。基因本体分析表明，三维培养的细胞中参与转录和翻译调控、细胞外基质组织和染色质重塑的生物学过程上调。三维培养条件还诱导了与Wnt和粘着斑信号相关的基因上调，而与p53信号通路相关的基因下调。我们的研究结果首次揭示了三维微环境中的机械信号转导刺激未经处理的胚胎干细胞自我更新的可能机制。
