In vitro-derived medium spiny neurons recapitulate human striatal development and complexity at single-cell resolution

• Published in: Cell reports methods Vol. 2; no. 12; p. 100367
• Main Authors: Conforti, Paola, Bocchi, Vittoria Dickinson, Campus, Ilaria, Scaramuzza, Linda, Galimberti, Maura, Lischetti, Tiziana, Talpo, Francesca, Pedrazzoli, Matteo, Murgia, Alessio, Ferrari, Ivan, Cordiglieri, Chiara, Fasciani, Alessandra, Arenas, Ernest, Felsenfeld, Dan, Biella, Gerardo, Besusso, Dario, Cattaneo, Elena
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19-12-2022; Elsevier
• Subjects: embryonic stem cells; human medium spiny projection neurons; Medicin och hälsovetenskap; midkine signaling; neuronal differentiation protocol; retinoic acid; single-cell RNA-seq; striatal development; midkine signaling; retinoic acid; embryonic stem cells; striatal development; single-cell RNA-seq; human medium spiny projection neurons; CP: stem cell; neuronal differentiation protocol
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2022.100367

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington’s disease (HD).
[Display omitted]
•An efficient method to generate D1 and D2-medium spiny neurons (MSNs) from hESC•The protocol shows high reproducibility between different batches•MSNs show significant similarity to human fetal MSNs at single-cell resolution•Modulating the midkine pathway enhances the number of MSNs generated
Although methods to develop medium spiny neurons (MSNs) have been reported, they are not able to reproduce D1- and D2-MSNs. Furthermore, all protocols use a restricted panel of genes to define MSNs, making it difficult to measure their authenticity. Finally, they all require extended periods of differentiation to obtain functional neurons. To solve these problems, we developed a protocol that enables the generation of functional D1- and D2-MSNs in just 25 days in culture. Single-cell RNA sequencing shows that these MSNs have significant resemblance with human fetal MSNs. Moreover, we define the midkine pathway as an important mediator of MSN differentiation.
Conforti et al. develop a protocol to generate authentic and functional D1 and D2-medium spiny neurons (MSNs). These MSNs show a significant resemblance to human fetal MSNs at single-cell resolution. By modulating the midkine pathway with retinoic acid, they show they can further increase the number of MSNs generated.