Three-dimensional culture of single embryonic stem-derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling

Abstract

In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES-NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES-NSPCs retained high cell viability and proliferated within small-sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII-tubulin and NF200 over time. At day 14, cell-matrix constructs mainly comprised NSPCs and neurons (46.5% βIII-tubulin cells). Gamma-aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES-NSPCs expressed matriptase and secreted MMP-2/9, with MMP-2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES-NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three-dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES-NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin-based hydrogels for ES-NSPC delivery into the injured central nervous system. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of Laminin Derived Peptides IKVAV and LRE Tethered to Hyaluronic Acid on hiPSC Derived Neural Stem Cell Morphology, Attachment and Neurite Extension

“…Laminin, fibronectin and collagen IV expression are associated with neural differentiation [46,47]. However, fibronectin is typically expressed at a higher concentration than the other two proteins [47]. Similar to a previous study [78], the availability of laminin signaling in the matrix reduced the intensity of fibronectin staining ( Figure 5).…”

“…MMP 9 expression was localized on the cell body on both surfaces (Figure 4). Neurons express fibronectin and collagen IV during differentiation [46,47]. Staining for fibronectin was found to be less intense on PEP-HA compared to DIFF-HA surfaces ( Figure 5), while collagen IV was not detected on either surface.…”

“…Differences in MMP activity can lead to changes in ECM content [20]. Laminin, fibronectin and collagen IV expression are associated with neural differentiation [46,47]. However, fibronectin is typically expressed at a higher concentration than the other two proteins [47].…”

Combining Stem Cells and Biomaterial Scaffolds for Constructing Tissues and Cell Delivery

“…3 [66]. Many groups have investigated a variety of fibrin hydrogel formulations for engineering neural tissue from stem cells, including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), in vitro and for delivering stem cells into the damaged nervous system in vivo [67][68][69][70][71][72][73][74][75][76][77]. Other groups have examined the behavior of MSCs seeded inside of fibrin clots and treated with growth factors for use in engineering bone.…”

Engineering geometrical 3-dimensional untethered in vitro neural tissue mimic

“…Seeding Cell Density Affects the Structure of the NTM. Previous works have studied the effects of changing the stiffness of the fibrin matrix on the resulting viability of tethered NTMs by changing the concentration of fibrinogen with respect to the added thrombin (21)(22)(23). We build upon those studies, but due to our desire to obtain mechanically stable and untethered functional 3D in vitro constructs, we studied the effect of varying the seeding cell concentration.…”