Nanofiber Matrices Promote the Neuronal Differentiation of Human Embryonic Stem Cell-Derived Neural Precursors In Vitro

Abstract

The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs.

Fabrication of poly (ϵ-caprolactone) microfiber scaffolds with varying topography and mechanical properties for stem cell-based tissue engineering applications

“…A similar body of work demonstrates the role of physical cues presented by scaffolds such as elasticity, micro and nanostructures of these structures can influence stem cell differentiation as well [9,[36][37][38]. For example, aligned nanoscale topography significantly enhanced the neuronal differentiation of ESCs [9,39]. For electrospun fibers, these physical and mechanical factors include morphological and mechanical properties of such scaffolds, which are highly influenced by altering fiber diameter.…”

Neural differentiation from pluripotent stem cells: The role of natural and synthetic extracellular matrix

“…The contact guidance and topography effects of 3-D scaffolds on neural differentiation were revealed in several studies recently [77,83] . The 3-D microfibrous poly(ethylene terephthalate) (PET) scaffolds have been shown to support neural differentiation of PSCs induced in an astrocyte-conditioned medium [84,85] .…”

“…Different 3-D synthetic ECM scaffolds including hydrogels, microfibrous, and nanofibrous matrices have been used for neural differentiation from PSCs or PSC-derived neural precursors (Table 4) [75][76][77][78] . For example, using chitin-alginate 3-D microfibrous scaffolds together with RA and noggin, nestin-expressing neural progenitors were derived from three independent hiPSC and hESC lines [75] .…”

“…The 3-D differentiation in PET scaffolds was also demonstrated in stirred bioreactors for potential scale up [85] . The effects of fiber diameter and fiber orientation of polycaprolactone fiber matrices were evaluated for hESC-derived neural precursors [77] . The NPCs adhered on the aligned fibers showed a higher rate of neuronal differentiation as compared to cells cultured on random micro-and nano-fibers (62%-86% vs 27%-32%).…”

Electrospun biomaterial scaffolds with varied topographies for neuronal differentiation of human‐induced pluripotent stem cells

“…The same study reported that mouse ESC-derived neurons seeded on the aligned fibers showed longer neurite outgrowth compared to the same cells seeded on the randomly-oriented nanofibers. Other researchers have shown that seeding human ESCderived neural cells onto aligned nanofibers enhanced neuronal differentiation compared to randomly orientated nanofibers [26]. Additionally, small molecules and growth factors can be encapsulated inside of such nanofiber scaffolds [24,[27][28][29].…”