US 12,084,679 B2
Methods of in vitro differentiation of midbrain dopamine (mDA) neurons
Lorenz Studer, New York, NY (US); Stefan Irion, New York, NY (US); Mark Tomishima, Chatham, NJ (US); and Sonja Kriks, San Francisco, CA (US)
Assigned to MEMORIAL SLOAN-KETTERING CANCER CENTER, New York, NY (US)
Filed by MEMORIAL SLOAN-KETTERING CANCER CENTER, New York, NY (US)
Filed on Nov. 9, 2020, as Appl. No. 17/093,126.
Application 17/093,126 is a continuation of application No. 15/820,941, filed on Nov. 22, 2017, granted, now 10,858,625.
Application 15/820,941 is a continuation of application No. PCT/US2016/035312, filed on Jun. 1, 2016.
Claims priority of provisional application 62/169,379, filed on Jun. 1, 2015.
Claims priority of provisional application 62/169,444, filed on Jun. 1, 2015.
Prior Publication US 2021/0123018 A1, Apr. 29, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 5/0793 (2010.01); A61K 35/30 (2015.01); A61K 35/545 (2015.01); A61P 25/16 (2006.01)
CPC C12N 5/0619 (2013.01) [A61K 35/30 (2013.01); A61K 35/545 (2013.01); A61P 25/16 (2018.01); C12N 2501/13 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/41 (2013.01); C12N 2501/415 (2013.01); C12N 2501/727 (2013.01); C12N 2506/02 (2013.01)] 27 Claims
 
1. An in vitro method for differentiating pluripotent stem cells comprising:
(a) contacting a plurality of pluripotent stem cells with at least one inhibitor of TGFβ/Activin-Nodal signaling; and
(b) contacting the plurality of pluripotent cells with at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling to obtain a population of differentiated cells expressing forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A), wherein the concentration of the at least one activator of Wnt signaling is increased during its contact with the plurality of pluripotent cells, and
wherein i) the concentration increase is initiated between about 2 days and about 6 days from the initial contact of the at least one activator of Wnt signaling with the cells and ii) the concentration of the at least one activator of Wnt signaling is increased by between about 100% and about 2000% of the initial concentration of the at least one activator of Wnt signaling contacted with the cells.