US 12,084,680 B2
Methods for efficient derivation of human motor neurons from diverse spinal regions
Ethan Scott Lippmann, Nashville, TN (US); Neha Sehgal, Philadelphia, PA (US); and Randolph Scott Ashton, Madison, WI (US)
Assigned to Wisconsin Alumni Research Foundation, Madison, WI (US)
Filed by Wisconsin Alumni Research Foundation, Madison, WI (US)
Filed on Jan. 14, 2021, as Appl. No. 17/149,713.
Application 17/149,713 is a division of application No. 15/475,831, filed on Mar. 31, 2017, granted, now 10,920,193.
Claims priority of provisional application 62/317,115, filed on Apr. 1, 2016.
Prior Publication US 2021/0139849 A1, May 13, 2021
Int. Cl. C12N 5/0797 (2010.01)
CPC C12N 5/0623 (2013.01) [C12N 2500/38 (2013.01); C12N 2501/119 (2013.01); C12N 2501/415 (2013.01); C12N 2501/727 (2013.01); C12N 2501/999 (2013.01)] 6 Claims
 
1. A method of generating post-mitotic motor neurons having a specified spinal cord regional identity, comprising the steps of:
i. culturing an adherent monolayer of SOX2+ and Brachyury+ neuromesodermal progenitor cells in a neural differentiation base medium that comprises FGF and a first concentration of a Wnt/β-catenin signaling pathway agonist for about 4 days;
ii. transiently exposing the cultured cells of (i) to a second concentration of the Wnt/β-catenin signaling pathway agonist for about 6 hours whereby NKX6.1+ ventral progenitor cells are obtained, wherein the second concentration is at least two times greater than the first concentration;
iii. replacing the neural differentiation base medium comprising the second higher concentration of the Wnt/β-catenin signaling pathway agonist with a neural differentiation base medium supplemented with a retinoid and at least one sonic hedgehog (SHH) signaling pathway agonist and lacking any Wnt/β-catenin signaling pathway agonist;
iv. culturing the NKX6.1+ ventral progenitor cells in the neural differentiation base medium of (iii) for about 2 days whereby a cell population comprising at least 80% OLIG2+ motor neuron progenitors, at least 95% NKX6.1+ motor neuron progenitors, and at least 95% Pax6+ motor neuron progenitors is obtained about 6 days from the start of step (i); and
v. exposing the motor neuron progenitor cells to a retinoid and to at least one SHH signaling pathway agonist in the neural differentiation base medium, and optionally exposing the motor neuron progenitor cells to dorsomorphin (DM) in the base medium, until SMI32+, ISL1+, and HB9+ post-mitotic motor neurons having a specified spinal cord regional identity are obtained.