**2. Unaddressed problems in current motor neuron differentiation strategies using induced pluripotent stem cells (iPSCs)**

The development of induced pluripotent stem cells (iPSCs) has enabled researchers to study many *human diseases,* including congenital neurodegenerative diseases, from a developmental perspective. Established somatic reprogramming techniques, using only four transcription factors (Oct4, Sox2, Klf4, and c-Myc), enable scientist to generate patient-derived stem cell lines harboring specific patient mutations. Advances in genetic editing strategies have also facilitated the establishment of isogenic controls from these mutants and comparison of cells derived from these paired iPSC population allow analysis of how a specific patient mutation alters the phenotype of cells that otherwise harbor identical genotypes [19]. Despite their enormous potential to replace or augment animal disease models, current neuronal iPSC models come with critical shortcomings that have been a major roadblock for their wider adoption in biomedical applications. Although iPSCs show similar transcriptomic profiles with those of embryonic stem cells (ESC), especially for the genes governing pluripotency, even state-of-the-art differentiation strategies for generating human neurons from iPSCs are far from perfect in terms of their capacity to produce cells that accurately reflect their *in vivo* counterparts [20–23]. Populations obtained from 20 to 50 days of *in vitro* neuronal differentiation often contain a mixture of various unknown subtypes of neurons, and glial cells. Even cells from outside the neuroglial lineage are often observed, and these cells tend to outcompete the postmitotic neuronal populations over time [22–25]. Moreover, commonly used differentiation protocols often lack reproducibility, which can result in the production of transcriptionally and functionally inconsistent neuronal populations from one batch to the next. Thus, it is reasonable to posit that such shortcomings in iPSC differentiation methods will have a negative impact on the accuracy and reproducibility of downstream research results when iPSC-derived neurons are used as predictors of human neural responses to chemical or pathological challenges. In addition to the disparity between *in vivo* human neurons and iPSC-derived neurons, a more challenging issue lies in the fact that we do not know how to reliably promote the maturation of iPSC-derived neurons toward an adult phenotype, which is critical for the study of late-onset neurodegenerative disorders such as ALS and Parkinson's disease (PD) [18, 26–30].

Most current motor neuron differentiation protocols rely on small molecule treatments to sequentially induce ectoderm, neuroectoderm, ventral spinal neuron progenitors, and finally a mixture of premature motor neurons and interneurons. This is often then followed by treatment with trophic factors to induce further maturation of those early-stage neurons [31–34]. In the majority of these protocols, undifferentiated iPSCs are initially treated with dual-SMAD inhibitors (SB431542 and LDN193189) to inhibit TGF-beta and BMP signaling pathways, which drives pluripotent stem cells toward the ectoderm lineage. Du *et al*. found that additional treatment of CHIR99021, which turns on the Wnt pathway, significantly enriches early-stage cultures with proliferative neural progenitors and this, in turn, results

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

in the formation of a greater number of so-called "neural rosettes" around 10 days post-induction. A critically important small molecule that most protocols use to provide a caudalization cue to the developing neural progenitors is retinoic acid (RA) [35]. RA is a pivotal molecule regulating embryonic patterning and development. It is a metabolic product of vitamin A (retinol) synthesized by the paraxial mesoderm. RA mediates the expression of HOX genes that are sequentially activated during embryonic development and is responsible for regulating the regional patterning of neuronal subtypes vertically along the spinal cord (**Figure 3**). The timing of RA expression dictates when colinear HOX gene expression is stopped, thereby controlling the regional patterning of neurons as well as promoting the transition from neuromesoderm into neuroectoderm [36, 37]. Given the importance of RA physiology in spinal motor neuron development, it has been a major target of research to improve motor neuron differentiation protocols. However, our understanding of RA-mediated motor neuron differentiation has not progressed far beyond the initial findings regarding limited aspects of its mechanism of action, leaving an important knowledge gap in terms of our understanding of spinal motor neuron development.

Once a caudal phenotype is established by RA, sonic hedgehog (SHH) or its agonist (e.g., purmorphamine with or without SAG (Smo agonist) supplementation) is then typically used to derive progenitors toward a ventral spinal identity. SHH is a vertebrate homolog of the Drosophila protein hedgehog and is expressed by the

#### **Figure 3.**

*Schematic illustration of the spinal cord development in vivo. (A) In the early stages of development, a process called gastrulation occurs, which leads to the differentiation of cells in the inner cell mass (ICM) into three germ layers: Ectoderm, endoderm, and mesoderm. The dorsal part of the ectoderm undergoes further specialization into the neuroectoderm by inhibiting BMP and activin signaling while enhancing FGF and Wnt signaling, particularly in higher organisms. Neuralization progresses as a neural plate form and subsequently folds to create neural folds, which then fuse to form the neural tube. The neural tube is organized along the anterior-posterior axis (rostral-caudal) through the presence of a retinoic acid (RA) gradient, primarily regulated by Raldh2. RA plays a crucial role in establishing the initial boundaries between the spinal cord and hindbrain versus forebrain and hindbrain. Fgfs and Gdf11 counteract the effects of RA and contribute to the specification of more caudal spinal cord cell types. (B) the schematic illustrates key signaling factors that play a crucial role in organizing the anterior–posterior (rostral–caudal) and dorsal–ventral axes of the developing nervous system during embryonic development. It specifically focuses on a coronal section through the developing telencephalon. (fibroblast growth factor: FGF, bone morphogenetic protein: BMP, retinoic acid: RA, sonic hedgehog: Shh).*

notochord and floor plate in the developing spinal cord. It provides signals necessary for positional patterning in the spinal cord. The concentration gradient of SHH determines whether the unpatterned progenitor cells have dorsal or ventral identity during the course of spinal neuron development. Despite the present understanding and detailed knowledge on the spinal neuron development and the cues necessary to drive the adoption of a motor neuron phenotype, the cells obtained from these protocols typically result in the adoption of an embryonic or neonatal phenotype. The use of particular biomaterials that exhibit various functionality upon applications is one of the promising strategic methods to achieve greater levels of maturation in these cells *in vitro*. Motor neuron differentiation of iPSCs using such functional biomaterials to improve the physiological relevance *in vitro* culture is discussed in detail below.
