Progress and challenges in directing the differentiation of human iPSCs into spinal motor neurons

Abstract

Motor neuron (MN) diseases, including amyotrophic lateral sclerosis, progressive bulbar palsy, primary lateral sclerosis and spinal muscular atrophy, cause progressive paralysis and, in many cases, death. A better understanding of the molecular mechanisms of pathogenesis is urgently needed to identify more effective therapies. However, studying MNs has been extremely difficult because they are inaccessible in the spinal cord. Induced pluripotent stem cells (iPSCs) can generate a theoretically limitless number of MNs from a specific patient, making them powerful tools for studying MN diseases. However, to reach their potential, iPSCs need to be directed to efficiently differentiate into functional MNs. Here, we review the reported differentiation protocols for spinal MNs, including induction with small molecules, expression of lineage-specific transcription factors, 2-dimensional and 3-dimensional cultures, as well as the implementation of microfluidics devices and co-cultures with other cell types, including skeletal muscle. We will summarize the advantages and disadvantages of each strategy. In addition, we will provide insights into how to address some of the remaining challenges, including reproducibly obtaining mature and aged MNs.

Decellularized Brain Extracellular Matrix Hydrogel Aids the Formation of Human Spinal-Cord Organoids Recapitulating the Complex Three-Dimensional Organization

“…Subsequently, SAG was utilized to activate Sonic Hedgehog (SHH) receptors, facilitating the induction of the ventral spinal cord, which is also associated with the formation of caudate protrusions. ,, During the SAG treatment, the previously noted small hemispherical protrusions on the organoids’ surface progressively diminished, and the wrinkling index concurrently decreased (Figure F). Following this, induction of the dorsal spinal cord was executed using BMP-4. , During BMP-4 treatment, the organoid may undergo subtle changes in morphology (e.g., spherical, ellipsoidal, extended) (Figure B). Finally, media enriched with the Brain-Derived Neurotrophic Factor (BDNF) and Glial Cell Line-Derived Neurotrophic Factor (GDNF) were employed for the maintenance culture of spinal cord organoids (Figure A).…”

“…Following this, induction of the dorsal spinal cord was executed using BMP-4. 23,26 During BMP-4 treatment, the organoid may undergo subtle changes in morphology (e.g., spherical, ellipsoidal, extended) (Figure 3B). Finally, media enriched with the Brain-Derived Neurotrophic Factor (BDNF) and Glial Cell Line-Derived Neurotrophic Factor (GDNF) were employed for the maintenance culture of spinal cord organoids (Figure 3A).…”

Human Placenta Decellularized Extracellular Matrix Hydrogel Promotes the Generation of Human Spinal Cord Organoids with Dorsoventral Organization from Human Induced Pluripotent Stem Cells

“…We utilized the SMAD inhibitor SB431542 (SB) to reduce the transforming growth factor-β (TGF-β) activity, LDN193189 (LDN) to inhibit bone morphogenetic protein (BMP-4) signaling, and CHIR-99021 (CHIR) to block glycogen synthase kinase 3 (GSK3), thereby activating WNT signaling. 23 These steps, illustrated in Figure 3A, are grounded in the established research. By synergistically combining WNT signaling activation with classical dual SMAD inhibition, our method effectively directs hiPSC cell spheres away from endodermal and mesodermal differentiation pathways.…”

Induced Pluripotent Stem Cells and Their Applications in Amyotrophic Lateral Sclerosis

“…Direct differentiation of iPSCs into MNs is the first necessary step toward their subsequent applications. The differentiation protocols for spinal MNs include induction with small molecules, expression of lineage-specific transcription factors and 2-dimensional and 3-dimensional cultures, as well as the implementation of microfluidics devices and co-cultures with other cell types, including skeletal muscle [ 45 ]. All of the approaches have advantages and disadvantages.…”