Abstract
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.
Highlights
Embryonic stem cells (ESCs) are pluripotent cells capable of unlimited self-renewal and differentiation into all three germ layers [1,2], making them an attractive source for the development of in vitro disease models and drug discovery as well as tissue engineering and cellular therapies [3,4,5]
We investigated the 3-D culture of naïve human embryonic stem cells (ESCs) (Elf1 cells) using self-assembling scaffolds comprised of two synthetic 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate end groups (PEG-8-Acr). 3-D culture in the PEG-8SH/PEG-8-Acr scaffolds supported ESC self-renewal and pluripotency
We investigated the effect of 3D culture on the growth, maintenance, and gene expression of naïve human ESCs
Summary
Embryonic stem cells (ESCs) are pluripotent cells capable of unlimited self-renewal and differentiation into all three germ layers [1,2], making them an attractive source for the development of in vitro disease models and drug discovery as well as tissue engineering and cellular therapies [3,4,5] These applications require stringent conditions for characterization and expansion as well as differentiation of cells into homogenous populations of progenitors or derivatives. Mouse ESCs, isolated at an earlier developmental stage, show improved clonal growth and viability following single-cell dissociation and are capable of contributing to blastocyst chimeras [9] Based on their divergent characteristics in vitro, two distinct stages of pluripotency were proposed, an early naïve state (mouse ESCs) and a primed state (human ESCs and mouse EpiSCs) [10]
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