Transcriptomic analysis of naïve human embryonic stem cells cultured in three-dimensional PEG scaffolds

Derivation of primed and naive human embryonic stem cells (ESCs) have prompted an increased interest in devising culture conditions for maintaining their pluripotency and differential potential. Naive ESCs are characterized by improved viability, proliferation, and differentiation capacity in comparison to primed ESCs. However, traditional two-dimensional (2-D) cell culture techniques fail to mimic the three-dimensional (3-D) in vivo microenvironment, which results in altered morphological and molecular characteristics of ESCs. Here, we describe the use of 3-D self-assembling scaffolds that support growth and maintenance of the naive state characteristics of human ESC line, Elf1. Scaffolds were formed via a Michael addition reaction upon combination of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups. 3-D scaffolds not only maintained the naive state, but also supported long-term growth for up to 3 weeks without requiring routine passaging and manipulation. 3-D grown cells exhibited upregulation of core (OCT4, NANOG, and SOX2) and naive (KLF17, KLF4, TFCP2L1, DPPA3, and DNMT3L) genes. These genes returned to normal levels when 3-D grown cells were propagated under 2-D culture conditions. Examination of RNA-sequencing demonstrated significant changes in gene expression profiles between 2-D and 3-D grown Elf1 cells. Gene Ontology analysis revealed upregulation of biological processes involved in the regulation of transcription and translation, as well as {beta}-catenin-TCF complex assembly, extracellular matrix organization, and chromatin remodeling in 3-D grown Elf1 cells. 3-D culture conditions also induced upregulation of genes associated with several signaling pathways including Wnt signaling and focal adhesion. However, p53 signaling pathway associated genes were downregulated under these culture conditions. Our findings provide insight into the possible mechanisms of prolonged self-renewal as well as upregulation of pluripotent genes stimulated by the transduction of mechanical signals from the 3-D microenvironment.