US 12,247,219 B2
Method for inducing differentiation of corneal epithelial cells from pluripotent stem cells
Kohji Nishida, Suita (JP); Ryuhei Hayashi, Suita (JP); and Yuki Ishikawa, Suita (JP)
Assigned to Osaka University, Osaka (JP)
Filed by Osaka University, Suita (JP)
Filed on Jun. 15, 2021, as Appl. No. 17/348,174.
Application 17/348,174 is a continuation of application No. 15/542,200, granted, now 11,066,641, previously published as PCT/JP2016/050784, filed on Jan. 13, 2016.
Claims priority of application No. 2015-006074 (JP), filed on Jan. 15, 2015.
Prior Publication US 2021/0371818 A1, Dec. 2, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 5/079 (2010.01); C12N 5/074 (2010.01); C12N 5/10 (2006.01)
CPC C12N 5/0621 (2013.01) [C12N 5/0607 (2013.01); C12N 5/10 (2013.01); C12N 2500/90 (2013.01); C12N 2501/117 (2013.01); C12N 2501/727 (2013.01); C12N 2506/45 (2013.01)] 15 Claims
 
1. A method for producing a colony consisting of concentric circular-like zones of different ectodermal cell lineages of ocular cells, the method comprising:
subjecting human pluripotent stem cells to two-dimensional culture in a serum-free medium without using feeder cells to form the colony by autonomous differentiation of the human pluripotent stem cells,
wherein said subjecting comprises:
(a) seeding the human pluripotent stem cells in the serum-free growth medium in a culture vessel for a first period of at least 7 days, wherein the vessel is coated with an effective amount of laminin 511 E8 fragment and contains the serum-free growth medium, and
(b) replacing the growth medium in the culture vessel with a differentiation medium comprising serum replacement and culturing the seeded human pluripotent stem cells in the differentiation medium for a second period of at least 2 weeks to produce the colony comprising concentric circular-like zones of different ectodermal lineage cells,
wherein the concentric circular-like zones of different ectodermal cell lineages comprise a zone of neuroectodermal lineage cells, a zone of neural crest lineage cells/optic cup lineage cells, a zone of ocular surface ectodermal lineage cells, and a zone of surface ectodermal lineage cells, and
wherein within the zone of neuroectodermal lineage cells, the zone of neural crest lineage cells/optic cup lineage cells and the zone of ocular surface ectodermal lineage cells each comprises pax6-positive ectodermal lineage cells; each of the zone of ocular surface ectodermal lineage cells and the zone of surface ectodermal lineage cells comprises p63- and E-cadherin-positive ectodermal lineage cells; and wherein the zone of neuroectodermal lineage cells and the zone of neural crest lineage cells/optic cup lineage cells each comprises TUBB3-positive ectodermal lineage cells.