Differentiation of Neural Crest Stem Cells in Response to Matrix Stiffness and TGF-β1 in Vascular Regeneration

Abstract

The neural crest stem cells derived from human induced pluripotent stem cells (iPSC-NCSCs) are a valuable autologous cell source for tissue engineering and regenerative medicine. In this study, we investigated how iPSC-NCSCs could be regulated to regenerate arteries by microenvironmental factors, including the physical factor of matrix stiffness, and the chemical factor of transforming growth factor beta-1 (TGF-b1). We found that, compared to soft substrate, stiff substrate drove iPSC-NCSCs differentiation into smooth muscle cells, which was further enhanced by TGF-b1. To investigate the regulatory role of TGF-b1 in vivo, we fabricated vascular grafts composed of electrospun nanofibrous scaffolds, collagen gel, iPSC-NCSCs, and TGF-b1, and implanted them into athymic rats. The results showed that TGF-b1 significantly promoted extracellular matrix synthesis and increased mechanical strength of vascular grafts. This study presents a proof of concept that iPSC-NCSCs can be used as a promising autologous cell source for vascular regeneration when combined with physical and chemical engineering.

Updated perspectives on vascular cell specification and pluripotent stem cell-derived vascular organoids for studying vasculopathies

“…Nevertheless, protocol 3 was predominantly an in vitro differentiation strategy, where iPSCs are of mouse origin and the induction of VSMCs was not included; whereas protocol 1 encompassed both cyclic circumferential stretching and in vivo biological environment as two co-existing factors to induce VSMCs from human iPSCs. The induced VSMCs exhibited enhanced mechanical strength and more durable vascular grafts 23 . As for the growth factor combinations deployed for each protocol, there are no studies confirming which combination is the most optimal.…”

“…Based on key discoveries in embryonic vascular development, three wellaccepted methods have been established for the generation of vascular cells from hiPSCs (Figure 2): 1) hiPSCs were first differentiated into neural crest stem cells (NCSCs) by fibroblast growth factor 2 (FGF-2) treatment 21,22 , then matured into VSMCs by seeding the NCSCs onto a relative stiff matrix, which were maintained in serum-free media containing transforming growth factor-β1 (TGF-β1), as a result, the matured VSMCs exhibited enhanced mechanical properties 23 ; 2) hiPSCs were differentiated into mesoderm following the treatment of bone morphogenic protein 4 (BMP4) and GSK3 inhibitors CHIR99021/CP21, the mesoderm was then treated with vascular endothelial growth factor (VEGF) and Forskolin to induce VECs specification; alternatively, by using platelet-derived growth factor-BB (PDGF-BB) and Activin-A to induce VSMC specification 24 . As a result, the induced VECs and VSMCs were comparable to primary cells in transcriptomes, metabolomic profiles and functional properties, as well as drastically reduced differentiation period in contrast to previous study 25 ; 3) instead of traditional static culture set up, the introduction of dynamic forces, such as shear stress, combined with VEGF and FGF treatments, can accelerate VEC specification and yield a greater number of tube-like network; in particular, the improved maturation of VECs was accompanied by upregulated Notch1 signalling and F-Actin filament reaglinment by media flow 26 .…”

Manipulation of Stem Cells Fates: The Master and Multifaceted Roles of Biophysical Cues of Biomaterials

“…Most recent studies have focused on the simulation of ECM stiffness in 2D or 3D biomaterials. Commonly used hydrogels such as hyaluronic acid (HA), [ 23 ] polyacrylamide, [ 17b ] polyethylene glycol (PEG), [ 24 ] and poly l ‐lactide (PLLA), [ 25 ] as well as natural compounds such as collagen, [ 26 ] fibrin, [ 27 ] and peptide amphiphiles, [ 28 ] have been utilized to rebuild ECM‐like properties.…”

“…[ 14 ] Stiffer biomaterials promote differentiation of smooth muscle cells, while softer ones promote neural lineage differentiation, including neurons and neuroglia. [ 25 ] The aligned micro/nanostructure is another core element in the design of NTE biomaterials. For efficient neural signal transmission, the innate nerve has a hierarchical structure with highly ordered subunits, from a single axon to nerve fibers.…”

Building on a Solid Foundation: Adding Relevance and Reproducibility to Neurological Modeling Using Human Pluripotent Stem Cells

“…Addition of protein and small molecule patterning factors specifies regionality to these protocols to generate even more specialized cell types like dopaminergic (Kirkeby et al, 2012 ), hippocampal (Sarkar et al, 2018 ), serotonergic (Lu et al, 2016 ), and motor neurons (Hu and Zhang, 2009 ). Protocols for neural crest cell differentiation (Hackland et al, 2017 ; Tchieu et al, 2017 ) enable the generation of peripheral neurons (Prince et al, 1991 ) as well as their mesenchymal derivatives (cartilage, bone (Leung et al, 2016 ), fat (Gomez et al, 2019 ) and smooth muscle (Serrano et al, 2019; Delaney et al, 2020 ; Li X. et al, 2020 ). Even non-ectodermal contributors to the nervous system can be produced and incorporated into brain tissue models including microglia (Abud et al, 2017 ; McQuade et al, 2018 ), brain microvascular endothelial cells (Qian et al, 2017 ) and pericytes (Stebbins et al, 2019 ).…”