US 11,781,109 B2
Methods of producing bioengineered neuronal organoids (BENOs) and uses thereof
Wolfram-Hubertus Zimmermann, Goettingen (DE); and Maria Zafeiriou, Goettingen (DE)
Assigned to GEORGE-AUGUST-UNIVERSITAET GOETTINGEN STIFTUNG OEFFENLICHEN RECHTS, UNIVERSITAETSMEDIZIN, Goettingen (DE)
Appl. No. 16/621,707
Filed by Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts Universitätsmedizin, Goettingen (DE)
PCT Filed Jun. 8, 2018, PCT No. PCT/EP2018/065204
§ 371(c)(1), (2) Date Dec. 12, 2019,
PCT Pub. No. WO2018/228948, PCT Pub. Date Dec. 20, 2018.
Claims priority of application No. 17175874 (EP), filed on Jun. 13, 2017.
Prior Publication US 2020/0208105 A1, Jul. 2, 2020
Int. Cl. C12N 5/0793 (2010.01); C12N 5/079 (2010.01); G01N 33/50 (2006.01)
CPC C12N 5/0619 (2013.01) [C12N 5/0622 (2013.01); G01N 33/5058 (2013.01); C12N 2500/30 (2013.01); C12N 2500/90 (2013.01); C12N 2501/115 (2013.01); C12N 2501/15 (2013.01); C12N 2501/42 (2013.01); C12N 2503/02 (2013.01); C12N 2506/02 (2013.01); C12N 2506/45 (2013.01); C12N 2513/00 (2013.01); C12N 2533/54 (2013.01)] 10 Claims
 
1. A method for producing a bioengineered neuronal organoid (BENO) from pluripotent stem cells (PSCs), the method comprising:
(A) providing a source of PSCs;
(B) culturing the PSCs of step (A), wherein the PSCs are embedded in a matrix immersed in serum-free medium;
(C) culturing the PSCs in said matrix of step (B) in serum-free cell culture medium comprising a Rho-associated kinase inhibitor (ROCKi) and fibroblast growth factor-2 (FGF-2);
(D) culturing the forming BENO originating from the PSCs and the matrix of step (C) in serum free cell culture medium comprising retinoic acid and one or more inhibitors of Mothers against decapentaplegic homologue (SMAD) signaling to induce neurogenesis;
(E) culturing the forming BENO of step (D) in serum free cell culture medium comprising transforming growth factor-beta (TGF-beta) and FGF-2 to enhance genesis of stromal cells and neurogenesis; and
(F) culturing the forming BENO of step (E) in serum free cell culture medium comprising TGF-beta and one or more inhibitors of notch signaling to enhance genesis of stromal cells and neurodifferentiation,
wherein the matrix comprises collagen, and
wherein the BENO is produced within a three-dimensional (3D) environment, wherein the 3D environment is initially defined by the matrix.