US 12,467,917 B2
Reconstructing human early embryogenesis in vitro with pluripotent stem cells
Magdalena D. Zernicka-Goetz, Pasadena, CA (US); Berna Sozen, Pasadena, CA (US); and Victoria Jorgensen, Pasadena, CA (US)
Assigned to California Institute of Technology, Pasadena, CA (US)
Filed by California Institute of Technology, Pasadena, CA (US)
Filed on Mar. 11, 2022, as Appl. No. 17/692,790.
Claims priority of provisional application 63/161,935, filed on Mar. 16, 2021.
Prior Publication US 2022/0308041 A1, Sep. 29, 2022
Int. Cl. G01N 33/50 (2006.01); C12N 5/071 (2010.01); C12N 5/073 (2010.01); C12N 5/0735 (2010.01)
CPC G01N 33/5005 (2013.01) [C12N 5/0604 (2013.01); C12N 5/0606 (2013.01); C12N 5/0697 (2013.01); C12N 2310/14 (2013.01); C12N 2501/113 (2013.01); C12N 2501/115 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/415 (2013.01); C12N 2503/04 (2013.01); C12N 2506/45 (2013.01); C12N 2513/00 (2013.01)] 18 Claims
OG exemplary drawing
 
1. An in vitro method of generating an expanded pluripotency (EP) structure in three dimensions, the method comprising:
(a) contacting pluripotent stem cells (PSCs) with EP media and culturing the PSCs in the EP media to generate expanded pluripotent stem cells (EPSCs);
(b) contacting at least 5 EPSCs with a first substrate and a composition capable of supporting generation of an EP structure, wherein the composition comprises 20% to 30% EP media, 20% to 30% trophoblast stem cell (TSC) media, 45% to 55% in vitro fertilization (IVF) media, and an ALK5 kinase inhibitor, and wherein the composition further comprises FGF2 to facilitate cavitation of the EP structure and one or more of: a TGFβ ligand, a WNT agonist, and a ROCK inhibitor; and
(c) culturing the EPSCs in the composition for at least 4 days, and wherein step (c) comprises reducing the concentration of FGF2 in the composition by half and removing the ALK5 kinase inhibitor after at least 48 hours of culturing in the composition, wherein the EPSCs self-organize to generate the EP structure, wherein the EP structure comprises a single outside layer, an enlarged cavity, and an internal acentric compartment.