Fig 2: Phenotypic analysis of hFM-MSCs during the differentiation into MNs(A) Schematic representation of the key points of the motor neuron differentiation. The different phases of the differentiation steps and the small molecules used are reported in the timeline, whereas the specific MNs genes are indicated in red.(B) Morphological analysis of different differentiation steps. Black arrows indicate the clusterization of hFM-MSC-derived MNs. Magnification 10x, scale bar 100 µm. Pictures are representative of 5 independent experiments from 5 different biological samples.(C) EBs diameter. The graphs show the Mean ± SD (n = 3 *p <0.05, **p =0.001, ***p<0.001).(D) Gene expression of OCT4, PAX6, ISL1 and HB9 at different time points of the differentiation process, as indicated. Data are expressed as fold changes (Mean ± SD) respect to day 14. 18S was used as reference gene (n = 3, *p <0.05, **p =0.001, ***p<0.001). AA, Ascorbic acid. RA, retinoid acid. SAG, Smoothened receptor agonists. bFGF, basic Fibroblast growth factor . BDNF, Brain-derived neurotrophic factor. GDNF, Glial cell line-derived neurotrophic factor. CTNF, Ciliary neurotrophic factor. ßOH, ß-mercaptoethanol.

Fig 3: Neural ribbons preserve action potential firing properties(A) Illustrated timeframe. Differentiating SMN cultures were encapsulated into neural ribbons (NR) for 48 hr, then recovered and further matured with neurotrophic factors (NTFs) that were BDNF and GDNF before patch clamp analysis. Current clamp recordings were performed at two time points (days 32–35 and days 45-52) across N = 3-4 differentiating cultures per condition (‘n’ denotes total number of cells recorded, whereas ‘N’ represents the number of culture batches used for these experiments).(B) NR-encapsulated SMNs (left); adherent SMNs after collection from neural ribbons and subsequent culture (right images).(C) SMN hallmark biomarkers at the day of recording (day 35) by immunofluorescence. Scale bars for panels (C, D) are 50 µm.(D) Differentiating SMN cultures were encapsulated into neural ribbons for 48 hr, then recovered and further matured with BDNF and GDNF before obtaining whole-cell patch clamp recordings. The traces show the voltage responses to somatic current step injections (500 ms long) at d33-35 (left) and d45-52 (right). DC current injections were used to maintain the SMNs at -65 mV.(E) Characterization of passive and active membrane properties of different types of SMN cultures. The passive membrane properties included a measure of the resting membrane potential (Vrest), membrane resistance (Rm), and membrane capacitance (Cm). Vrest ranged between -36 mV at d33-35 and -46 mV at d32-34 with neural ribbons (*p = 0.023 by ANOVA). Rm differed between d32–34 and d51-52 only in the presence of NTFs (*p = 0.048). The active membrane properties included the rheobase, action potential threshold (APthr) and action potential peak (APpeak). Rheobase decreased at d32-34 after neural ribbon encapsulation, and was associated with a hyperpolarization of the AP threshold (*p = 0.047), which also occurred between d32–35 and d45-52 SMN groups (*p = 0.032). AP peak was reduced between these SMN groups (*p = 0.039). A similar effect was also detected between d32–34 and d51-52 cultures treated with growth factors (*p = 0.047). As a consequence, the AP amplitude was slightly reduced at d51-52 in the presence of growth factors (*p = 0.048).(F) f/I plots illustrating the firing output of SMNs in the experimental conditions described above, for 500 ms-long current step injections of increasing amplitude. The overall excitability of these SMNs did not show statistically significant differences across different types of culturing conditions. The recordings were obtained from N = 3-4 differentiating cultures per condition (‘n’ denotes total number of cells recorded, whereas ‘N’ represents the number of culture batches used for these experiments). Data represent mean ± SEM.