**3. The use of biomaterials to improve the physiological relevance of iPSC-derived human neurons**

Variety of biomaterials, as forms of hydrogels, nanoparticles, scaffolds and mesh, and so forth, have been recognized as attractive resources in the field of regenerative medicine and tissue engineering due to their physical and chemical tunability as well as their biocompatibility with most human stem cells (**Figure 4**). In particular, the development of biocompatible materials that can support functional tissue generation has gained significant interest in recent years.

For example, a hydrogel is a hydrophilic polymer resembling human tissue with high biocompatibility. Various monomeric compounds have been studied to form the hydrogel by modulating the degree of cross-linking to control the mechanical strength and releasing kinetics of embedded payloads such as growth factors [38]. Gelatin is one of the natural polymers that is a precursor of collagen. Both collagen and gelatin have high biocompatibility and low toxicity, but there are some issues regarding complex purification and modifying short degradation rates [39]. Both of them are often used to form an extracellular matrix (ECM). Also, serum albumin is a natural polymer used to construct ECM [40]. It has long a half-life compared to other natural polymers [40–42]. A carbon nanotube is also used to make ECM because it is a tunable material that possesses mechanical and electrical properies [43] and it is easy to modify its surface. Moreover, it has biocompatibility and high cell attachment. Those features can be adjusted to mimic ECM.

#### **Figure 4.**

*Characteristics of biomaterials in scaffolds. Each class of biomaterials has numerous advantages and disadvantages in terms of its suitability as an engineered neural scaffold.*

#### *Exploring the Potential for Biomaterials to Improve the Development of Spinal Motor Neurons… DOI: http://dx.doi.org/10.5772/intechopen.113275*

In addition to synthetic biomaterials, such as polyethylene glycol (PEG), polylactide-co-glycolic acid (PLGA) has been widely demonstrated to stem cell differentiation and tissue regeneration because of its tunable physical and chemical properties that provide beneficial effects to construct functional human tissues *in vivo*. The central hypothesis of applying such biomaterials to neural tissue engineering is that the physiological relevance of differentiating neurons will be readily improved by providing an extracellular microenvironment that more closely recapitulates the native spatiotemporal niche where these cells occupy in developing embryos. Specifically, researchers have attempted to recapitulate the extracellular microenvironment during neural development by taking into account factors such as the cell–matrix interaction, intracellular interactions within the given environment, the physical and mechanical characteristics of the matrix surrounding the neural tube, the topographical status of extracellular proteins, and the oxygen and nutrient provision capabilities of the matrix. In the following section, we summarized the advances and limitations of the biomaterials that have been used so far to recreate the surrounding environment of nervous tissue during the developmental stages. 3D scaffolds are widely used as a means of structural cues that are responsible for physical and mechanical properties of tissue microenvironment such as matrix stiffness, adhesion, and migration during neuronal differentiation. On the other hand, micro- and nanoparticles are utilized to control the biological cues through spatiotemporal release of active ingredients to differentiate the neurons. Importantly, we discuss ideas that may help to recapitulate the two critical characteristics of native spinal cord development: (1) the exquisite spatiotemporal control of inductive cues secreted around the neural tube, which are the main drivers of neuronal differentiation and specialization throughout the course of normal spinal cord development and (2) the dramatic conversion from flattened neural plate to 3D neural tube during early phases of spinal cord development. We believe that these issues constitute a root cause of the disparity between spinal neurons developed *in vitro* versus *in vivo*, and addressing these issues may help disentangle the remaining issues still plaguing iPSC-derived neuronal development *in vitro*.

#### **4. 3D scaffolds: providing structural cues to iPSCs differentiation neurons**

Conventional cell culture experiments, conducted on flat plastic surfaces, have provided us with a wealth of basic cell biology knowledge for decades. However, 2D culture schemes do not accurately recreate the physical cues, such as matrix stiffness, topology, and interaction between cells and matrix, which are present in the extracellular environment during native 3D tissue development *in vivo*. Additionally, neural differentiation on 2D surfaces often suffers from issues caused by neural sheet delamination, which results in poor longevity of cultured cell populations and batchto-batch inconsistencies in differentiated populations. This becomes even more of a critical issue when it comes to generating multicellular tissues such as spinal cord that are required to have a continuous intracellular interactions and consistent delivery of differentiating cues from the surrounding microenvironments [44]. To address this issue, various biomaterials have been used to generate 3D scaffolds that provide a physiologically similar, material-permeable environment for exchanging morphogens, oxygen, and nutrients essential for each step of tissue development. Moreover, many studies have shown that 3D scaffold-mediated neural differentiation enables more complex interactions to develop between neural precursors and between cells

and ECM proteins. Furthermore, such scaffolds provide better spatial organization of cells, which mimics the native environment during neurogenesis more closely.

Scaffolds can come in various shapes and geometries, such as fibrillar morphologies (3D structures composed of long, fibrous protein chains) with respect to length, thickness, surface structure (smooth vs. rugged), and overall shape (straight vs. curly), which can be modified to impact cell-to-cell and cell-to-matrix interactions. There are three categories of scaffolds to address the structural cues: surface property, mechanical property, and electrical property (**Table 1**). The surface properties of scaffolds can often be changed by modulating pH or using surfactants to affect material properties such as water uptake, compressive moduli, and cross-linking.


#### **Table 1.**

*Structural cues derived by 3D scaffolds used in neuronal differentiation.*

*Exploring the Potential for Biomaterials to Improve the Development of Spinal Motor Neurons… DOI: http://dx.doi.org/10.5772/intechopen.113275*

Modulation of any of these properties could potentially affect the modality of neural differentiation within such structures. Therefore, it has been important to find a scaffold condition that promotes neurodevelopment that includes robust formation of axons and dendrites as well as the expression of neuron-specific proteins for synaptic transmission and electrophysiological function.

#### **Figure 5.**

*Currently studied applications of scaffolds in neuronal differentiation. (A) An example of surface property modulation. Carbon nanotube (CNT) network patterns are utilized to achieve selective growth and polarizationcontrolled neuronal differentiation of human neural stem cells (hNSCs). CNT roughness promotes the adhesion and longevity of primary neurons. This CNT pattern induces the selective growth and differentiation of hNMSs. Also, the CNT pattern is able to maintain cell-to-cell interaction well. As a result, the CNT pattern is a more stable and versatile platform, with optimal nano-topography and biocompatibility, with which to regulate hNSC growth in vitro [46]. (B) An example of mechanical property modification. Surfactant templating is shown to effectively tune the water uptake and compressive modulus of photo-cross-linked chitosan hydrogels, mitigating property fluctuations with changing pH, enabling greater attachment of iPSCs, and enhancing neuronal differentiation [49]. (C) An example of electrical property modulation. Conductive scaffolds incorporating topographical, biochemical, and electrical stimuli support attachment, proliferation, neuronal differentiation, and maturation of iPSCs, demonstrating their potential for nerve regeneration strategies without the need for growth factor supplementation [42].*

#### *Motor Neurons – New Insights*

For the use of biomaterials in neural tissue generation and remodeling, there are preconditions to be satisfied. Scaffolds should have the ability to provide structural support that is tailored to the needs of the embedded stem cells (in terms of stiffness etc.) and provide correct guidance cues to the developing neurons by allowing small molecules in the medium to reach the entire population of cells at desired concentrations and kinetics during tissue formation. Although challenging, researchers have been trying to achieve this goal by modulating the structural parameters of scaffolds such as the pore size, porosity (% of void area relative to the entire surface area of the scaffold), and surface stiffness. Additionally, work has been performed that explores the binding of functional groups on to the scaffold surface to induce biochemical interactions between the matrix and the embedded cells [54, 55]. For example, adjusting surface stiffness and wettability have been shown to impact the number of neurites growing out of individual cells and the number of neuritic branches that develop from these projections, which are both major metrics for assessing neuronal differentiation and maturation in culture (**Figure 5**) [46, 52, 53, 56, 57]. The mechanical properties of the substrate and the interaction between the matrix structure and cells are also important factors to consider [40, 58–62]. Nanocomposite materials composed of synthetic polymers and fillers allow bulk and local modulation of physical properties to create a high surface area to volume ratio and control interfacial binding strength, which can enable the release of neurotrophic factors that induce neuronal differentiation [62–65]. Lastly, electrical properties of the scaffold, such as conductive nanostructures like silver, gold, and carbon, can affect cell adhesion, migration, and orientation by electrical stimulation, which can also impact neuronal differentiation [62, 66, 67].
