	US 12,110,510 B2
	Functional cortico-spinal-muscle assembled spheroids
Jimena Andersen, Palo Alto, CA (US); and Sergiu P. Pasca, Palo Alto, CA (US)
Assigned to The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
Appl. No. 17/253,038
Filed by The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
PCT Filed Jun. 20, 2019, PCT No. PCT/US2019/038307 § 371(c)(1), (2) Date Dec. 16, 2020, PCT Pub. No. WO2019/246436, PCT Pub. Date Dec. 26, 2019.
Claims priority of provisional application 62/688,924, filed on Jun. 22, 2018.
Prior Publication US 2021/0261924 A1, Aug. 26, 2021
Int. Cl. C12N 5/071 (2010.01); C12N 5/077 (2010.01); C12N 5/0793 (2010.01); G01N 33/50 (2006.01)

CPC C12N 5/0697 (2013.01) [C12N 5/0619 (2013.01); C12N 5/0658 (2013.01); G01N 33/5082 (2013.01); C12N 2500/38 (2013.01); C12N 2501/01 (2013.01); C12N 2501/105 (2013.01); C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2501/13 (2013.01); C12N 2501/15 (2013.01); C12N 2501/41 (2013.01); C12N 2501/734 (2013.01); C12N 2501/999 (2013.01); C12N 2502/081 (2013.01); C12N 2502/1335 (2013.01); C12N 2506/45 (2013.01); C12N 2513/00 (2013.01)]

17 Claims

1. A method for producing functionally integrated human cortico-spinal-muscle assembled spheroids in vitro, the method comprising:

(i) inducing in a human pluripotent stem cell suspension culture a neural fate to provide a first spheroid of neural progenitor cells and a second spheroid of neural progenitor cells;

(ii) differentiating the neural progenitor cells in the first spheroid to differentiate into human spinal cord spheroids (hSC), and culturing the hSC with human skeletal muscle cells (hSkM) under integrating conditions to generate hSC-hSkM spheroids;

(iii) differentiating the neural progenitor cells in the second spheroid to differentiate into human cortical spheroids (hCS);

(iv) culturing the hCS and hSC-hSkM under conditions permissive for spheroid fusion while maintaining for an extended period of time in neural medium; wherein a functionally integrated human cortico-spinal-muscle assembled spheroid is differentiated comprising interacting glutamatergic projection neurons, motor neurons, interneurons, and muscle cells.