Controlling the Regional Identity of hPSC-Derived Neurons to Uncover Neuronal Subtype Specificity of Neurological Disease Phenotypes

Abstract

SummaryThe CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs.

Neural patterning of human induced pluripotent stem cells in 3-D cultures for studying biomolecule-directed differential cellular responses

“…RA was not included to minimize caudalization effect; (4) FGF-2 (10 ng/mL), purmorphamine (1 µM, Sigma) and RA (2 µM); (5) FGF-2, purmorphamine, and RA plus CHIR99021 (3 µM) [30]. After another 7-10 days in suspension, the 3-D neural progenitor cell (NPC) aggregates (day [15][16][17][18] were replated onto Geltrex-coated surface and characterized at day 20. For long-term characterizations at days 35-55, the neural progenitors were matured with brain-derived neurotrophic factor (10 ng/mL, R&D Systems) and gently passaged with trypsin once in two or three weeks with minimal pipetting.…”

“…Generating multiple neuronal subtypes from hiPSCs with a tunable differentiation protocol to delineate differential cellular responses is in a critical medical need [7,10,14,15].…”

Induced Pluripotent Stem Cell Technology in Regenerative Medicine and Disease Modeling

“…Therefore, it is difficult to directly compare different neuronal subtypes, even when they are derived from the same iPSCs. To establish a robust method to control the regional identity of PSC-derived neural progenitors and neurons based on a single protocol, Wnt, retinoic acid, and sonic hedgehog signaling was modulated to manipulate the anteroposterior and dorsoventral identities of neural progenitors 22) . Regional identity was maintained throughout neural differentiation, generating a variety of neuronal subtypes.…”

Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation

“…Due to the neural patterning effect, Wnt signaling can efficiently promote motor neuron differentiation from hPSCs, in combination with caudalization factor retinoic acid (RA) and ventralization factor SHH. 20,21 Wnt signaling may elevate the threshold level of SHH signaling to enrich motor neural progenitors. Using dual SMAD inhibition in the presence of Wnt activator CHIR99021, high purity of motor neural progenitors (>95% Oligo2 C ) can be generated in 12 days.…”