US 11,981,918 B2
Differentiation technique to generate dopaminergic neurons from induced pluripotent stem cells
Alexander Laperle, North Hollywood, CA (US); Samuel Sances, Santa Monica, CA (US); Nur Yucer, Los Angeles, CA (US); and Clive N. Svendsen, Pacific Palisades, CA (US)
Assigned to Cedars-Sinai Medical Center, Los Angeles, CA (US)
Appl. No. 17/041,788
Filed by CEDARS-SINAI MEDICAL CENTER, Los Angeles, CA (US)
PCT Filed Apr. 5, 2019, PCT No. PCT/US2019/026183
§ 371(c)(1), (2) Date Sep. 25, 2020,
PCT Pub. No. WO2019/195800, PCT Pub. Date Oct. 10, 2019.
Claims priority of provisional application 62/816,785, filed on Mar. 11, 2019.
Claims priority of provisional application 62/816,795, filed on Mar. 11, 2019.
Claims priority of provisional application 62/755,282, filed on Nov. 2, 2018.
Claims priority of provisional application 62/755,365, filed on Nov. 2, 2018.
Claims priority of provisional application 62/664,942, filed on May 1, 2018.
Claims priority of provisional application 62/664,827, filed on Apr. 30, 2018.
Claims priority of provisional application 62/664,888, filed on Apr. 30, 2018.
Claims priority of provisional application 62/653,697, filed on Apr. 6, 2018.
Prior Publication US 2021/0024886 A1, Jan. 28, 2021
Int. Cl. C12N 5/0793 (2010.01)
CPC C12N 5/0619 (2013.01) [C12N 2501/119 (2013.01); C12N 2501/13 (2013.01); C12N 2501/15 (2013.01); C12N 2501/41 (2013.01); C12N 2501/999 (2013.01); C12N 2506/45 (2013.01)] 14 Claims
 
1. A method, comprising:
providing a quantity of blood cell-derived induced pluripotent stem cells (iPSCs);
starting on day 0, culturing the iPSCs in the presence of a transforming growth factor (TGF)-beta inhibitor and an activin receptor-like kinase (MK) inhibitor for about 3 days;
following the about 3 days further culturing in the presence of the TGF-beta inhibitor, the AUK inhibitor, a Smoothened agonist, a RHO Kinase (ROCK) inhibitor and at least two growth factors for about 4 days;
following the about 4 days, additionally culturing in the presence of the TGF-beta inhibitor, the ROCK inhibitor, and retinoic acid and in the absence of the ALK inhibitor for about 4 days; and
continuing to culture in the presence of at least three additional growth factors and in the absence of the retinoic acid for at least 3 days.
 
11. A quantity of neurons made by a method comprising:
providing a quantity of blood cell-derived induced pluripotent stem cells (iPSCs);
starting on day 0, culturing the iPSCs in the presence of a transforming growth factor (TGF)-beta inhibitor and an activin receptor-like kinase (ALK) inhibitor for about 3 days;
following the about 3 days, further culturing in the presence of the TGF-beta inhibitor, the ALK inhibitor, a Smoothened agonist, a RHO Kinase (ROCK) inhibitor and at least two growth factors for about 4 days; following the about 4 days, additionally culturing in the presence of the TGF-beta inhibitor, the ROCK inhibitor, and retinoic acid and in the absence of the ALK inhibitor for about 4 days;
following the about 4 days of the additionally culturing in the presence of the TGF-beta inhibitor, the ROCK inhibitor, and retinoic acid and in the absence of the ALK inhibitor, continuing to culture in the presence of at least three additional growth factors and in the absence of the retinoic acid for at least 3 days; and
following the at least 3 days, continuing to culture in a maturation media including the at least three additional growth factors and lacking the ROCK inhibitor to generate dopamine producing neurons Gel-fs in about 30 days from day 0.
 
14. A method, comprising:
starting on day 0, culturing induced pluripotent stem cells (iPSCs) in the presence of LDN-193189 and SB431542 and in the absence of purmorphamine, sonic hedgehog, CHIR99012, and fibroblast growth factor 8 for about 3 days;
further culturing in the presence of LDS-193189, SB431542, purmorphamine, sonic hedgehog, CHIR99012, and fibroblast growth factor 8 for about 4 days;
additionally culturing in the presence of LDN-193189, CHIR99012 and retinoic acid for about 4 days;
continuing to culture in the presence of L-Ascorbic Acid, brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), c-AMP, TGF-Beta 3 and CHIR99012 for at least 3 days; and
continuing to culture in a maturation media including L-Ascorbic Acid, BDNF, GDNF, c-AMP, and TGF-Beta 3 and in the absence of CHIR99012 until about 30 days from day 0.