Transcriptomic Analysis of Naïve Human Embryonic Stem Cells Cultured in Three-Dimensional PEG Scaffolds

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.

Human primitive mesenchymal stem cell-derived retinal progenitor cells improved neuroprotection, neurogenesis, and vision in rd12 mouse model of retinitis pigmentosa

“…RNA-seq analysis was performed as previously described [ 38 ]. In brief, the total RNA of the samples was isolated using the GeneJet RNA purification kit (Thermo Fisher Scientific).…”

WNT and VEGF/PDGF signaling regulate self-renewal in primitive mesenchymal stem cells

“…The total RNA from LP and HP pMSCs cultured in FM and XM were isolated and sequenced by following the previously published protocol (29). Brie y, the RNA was quanti ed and quali ed using Agilent2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and Qubit Assay (Life Technologies).…”

“…In addition, MSCs exhibit anti-in ammatory and immunomodulation effects and home to the site of injury (9). They are preferable to embryonic stem cells (ESCs), which have ethical concerns and can cause teratomas (10,11). However, unlike ESCs, MSCs grow poorly and gradually lose self-renewal capability upon passaging, particularly in the fetal bovine serum medium (FM) (12).…”

Multiple Cell Cultures for MRI Analysis

“…Additionally, in their latest research, McKee and his group analyzed naive human embryonic stem cells (ESCs) grown on PEG-based 3D scaffolds. Based on the research, the possible mechanisms of self-renewal of naive ESCs were presented for the first time [ 158 ]. On the other hand, viscoelastic hydrogels enable the regulation of stress relaxation, which, as it turns out, after studies by Nam et al, has an important role in cell differentiation, spread, and proliferation.…”