US 12,378,527 B2
Differentiation of pluripotent stem cells using laminins LN-511, LN-521 and LN-221
Karl Tryggvason, Singapore (SG); Yan Wen Yap, Singapore (SG); Sun Yi, Sundyberg (SE); and Kristian Tryggvason, Sundyberg (SE)
Assigned to National University of Singapore, Singapore (SG); and BioLamina AB, Sundyberg (SE)
Filed by National University of Singapore, Singapore (SG); and BioLamina AB, Sundyberg (SE)
Filed on Apr. 9, 2021, as Appl. No. 17/226,611.
Application 17/226,611 is a continuation of application No. 14/895,669, granted, now 11,001,807, previously published as PCT/IB2014/002289, filed on Jul. 2, 2014.
Claims priority of provisional application 61/842,241, filed on Jul. 2, 2013.
Prior Publication US 2021/0301257 A1, Sep. 30, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 5/077 (2010.01); C12Q 1/6876 (2018.01)
CPC C12N 5/0657 (2013.01) [C12Q 1/6876 (2013.01); C12N 2501/115 (2013.01); C12N 2501/155 (2013.01); C12N 2501/16 (2013.01); C12N 2501/33 (2013.01); C12N 2501/415 (2013.01); C12N 2501/727 (2013.01); C12N 2501/998 (2013.01); C12N 2506/02 (2013.01); C12N 2533/52 (2013.01); C12Q 2600/158 (2013.01)] 8 Claims
 
1. A method for obtaining cardiomyocyte progenitor cells from human pluripotent stem cells, comprising:
(a) seeding the human pluripotent stem cells onto a substrate, wherein the substrate comprises a mixture of two laminins consisting of (i) recombinant laminin-521 (LN-521) or recombinant laminin-511 (LN-511) and (ii) recombinant laminin-221 (LN-221);
(b) culturing the human pluripotent stem cells on the substrate in a differentiation medium containing a GSK-3 inhibitor to stimulate Wnt signaling to obtain brachyury positive cells;
(c) culturing the brachyury positive cells on the substrate in a differentiation medium containing a Wnt inhibitor to inhibit Wnt signaling; and
(d) removing the differentiation medium containing the Wnt inhibitor from step (c) and replacing with a basal medium to obtain cardiomyocyte progenitor cells that express Islet-1 and NKX2.5.