Abstract
For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell—material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.
Highlights
Unlike peripheral nervous system (PNS), neurons in the central nervous system (CNS) do not spontaneously regenerate injured axons because of extrinsic inhibitory factors and intrinsically lower growth capacity [1,2]
The cell culture medium for ST1 cells was composed of Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich) supplemented with 20% (v/v) fetal bovine serum (FBS; Biochrom AB), 1mM L-glutamine, 1% nonessential amino acids (NEAA; Gibco, Life Technologies), 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol (Sigma Chemical), 1000 units/ml recombinant leukemia inhibitory factor (LIF; Chemicon, Millipore), 50 μg/ml penicillin, and 50 μg/ml streptomycin. induced pluripotent stem cell (iPSC) were maintained in the same self-renewal media used for ST1, except that iPSC medium was supplemented with 15% FBS
In contrast to previously reported neural differentiation protocols which typically rely on the addition of exogenous soluble factors, this study demonstrates an alternative method using Ncadherin-based biomimetic substrate to promote the differentiation of embryonic stem cell (ESC)- and iPSC-derived neural progenitor cells (NPCs)
Summary
Unlike peripheral nervous system (PNS), neurons in the central nervous system (CNS) do not spontaneously regenerate injured axons because of extrinsic inhibitory factors and intrinsically lower growth capacity [1,2]. Conditioning neurons by neural extracellular matrix (ECM) components and cell adhesion molecules (CAMs) are thought to play an important role in increasing the intrinsic growth capacity of neurons and neurites both in vitro and in vivo [3,4]. The extracellular part of neural CAM (N-cadherin) typically mediates calcium-dependent homophilic interaction and modulates several signaling pathways including Akt, Wnt/β-catenin, fibroblast growth factor (FGF)-2, and Rho GTPases [8,9,10,11,12]. In a number of studies, molecular tethering of CAMs and growth factors (GFs) has been proposed to understand key developmental signaling pathways by increasing protein stability, promoting persistent signaling, and reducing complexities associated with in vivo microenvironment [19,20,21,22]. Despite the emphasis given to biological surface modification in order to mimic pluripotent stem cell microenvironment, few studies have utilized these modified surface for controlling stem cell differentiation in a spatially defined and substrate-dependent manner
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
