## 4 Discussion
Expression of the oligodendrocyte transcription factor 2 (Olig2) was also quantified at this time point, and the overall expression for most groups was higher in comparison, with \(\beta\)T-III. N+ and PR+ showing the highest levels of expression (\(>\)60%), suggesting that either combination of small molecules (CHIR, SB, and LDN for N+) or (pure and RA for PR\(-\)) have a similar impact on the differentiation of NPCs into Olig2+ cells. These levels of \(\beta\)T-III and Olig2 expression are significantly higher than that reported by [PERSON] et al., where hiPSC-derived NPCs were bioprinted and differentiated, with a maximum gene expression of \(\beta\)T-III of \(\sim\) 2% and 15% for Olig2 after three weeks of differentiation [34]. The expression of the mature MN marker, HB9, at this time point was highly expressed for all groups at \(>\)61%. The highest level of HB9 expression was observed in the R+ group, however, the only statistically significant differences were observed between N+ and R+. Interestingly, P+, PR+, and PR\(-\) showed very similar levels of expression, suggesting that the presence of pure, alone or in combination with RA, is crucial for differentiating HB9+ neurons from NPCs using our novel bioink. However, given that PR+ and PR\(-\) have similar levels of HB9 expression with a yield of \(\sim\)76%, either treatment could be used for future analysis. However, R+ with the combination or RA and CHIR, SB, LDN is the most efficient, showing 99% yield of HB9+ MNs after 15 days. Our 3D bioprinted tissues were cultured for up to 30 days in vitro for further maturation, and characterization of MNs compared with previous studies where ICC staining was performed after seven days of culture [33]. Nonetheless, [PERSON] et al. cultured their bioprinted constructs for up to 40 days, showing the expression of mature neural markers, such as microtubule associated protein 2 (MAP2), gamma-aminobutyric acid GABA), and Synaptophysin [35]. ICC was performed for \(\beta\)T-III,the astrocyte marker (GFAP), and choline acetyl transferase (ChaT) to assess neuronal and glial cell differentiation. All of the groups showed \(\beta\)T-III expression; interestingly, P+ and PR+ self-aggregated into a spheroid structure as previously observed [35]. At this time point, all the scaffolds had been degraded, demonstrating that our bioink supported the cellular secretion of the required extra cellular matrix (ECM) components to maintain a 3D structure. GFAP expression was positive for N, R+, PR+, and PR-. ChaT expression was positive for N+, PR-, and PR+. Interestingly, PR+ showed the most cell aggregation and expression of \(\beta\)T-III and ChaT, and the lowest expression for GFAP, suggesting that the combination of these small molecules provides a better environment for MN differentiation and maturation. Further maturation and characterization of the tissues will be necessary in order to fully characterize their maturity and electrophysiological characteristics. These bioprinted neural tissues can be used as a complementary pre-clinical model to assess drug safety and toxicity in the early development of clinical trials. hiPSCs can be derived from the patient and matured into neural progenitor cells in order to perform such an analysis with patient specific cells. In the future, drug releasing microspheres can be deposited along with the cell-suspension within the bioink, where each fiber contains the required drug-releasing microspheres to promote differentiation into different cell types. More than one type of cell can be differentiated within these bioprinted tissues in order to mimic a more defined micro-architecture of the SC tissue along with a blood spinal cord barrier (BSCB).