	US 12,351,822 B2
	Stem cell culture systems for columnar epithelial stem cells, and uses related thereto
Wa Xian, Sugar Land, TX (US); Frank McKeon, Sugar Land, TX (US); and Matthew P. Vincent, Amesbury, MA (US)
Assigned to University of Houston System, Houston, TX (US)
Filed by UNIVERSITY OF HOUSTON SYSTEM, Houston, TX (US); and TRACT PHARMACEUTICALS, INC., West Hartford, CT (US)
Filed on Jun. 19, 2023, as Appl. No. 18/337,292.
Application 18/337,292 is a division of application No. 16/958,074, granted, now 11,725,185, previously published as PCT/US2018/067858, filed on Dec. 28, 2018.
Claims priority of provisional application 62/724,937, filed on Aug. 30, 2018.
Claims priority of provisional application 62/611,176, filed on Dec. 28, 2017.
Prior Publication US 2023/0357714 A1, Nov. 9, 2023
Int. Cl. C12N 5/074 (2010.01)

CPC C12N 5/0607 (2013.01) [C12N 2500/38 (2013.01); C12N 2501/105 (2013.01); C12N 2501/135 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/165 (2013.01); C12N 2501/33 (2013.01); C12N 2501/415 (2013.01); C12N 2501/603 (2013.01); C12N 2501/727 (2013.01)]

12 Claims

1. A method for isolating a stem cell from columnar epithelial tissue comprising:

(i) culturing dissociated epithelial cells from a columnar epithelial tissue sample to form stem cell colonies, wherein the dissociated cells and cell colonies are cultured in a medium comprising a ROCK (Rho Kinase) inhibitor, a Wnt agonist, a mitogenic growth factor, insulin (or an insulin mimetic) or IGF, a BRAF inhibitor, a VEGF inhibitor, nicotinamide, a Notch Agonist, a TGFβ signaling pathway inhibitor, and a Bone Morphogenetic Protein (BMP) antagonist;

(ii) isolating single stem cells from the cell colonies, and

(iii) culturing isolated single stem cells from step (ii) individually to form cultures purified stem cell clones;

wherein each of the stem cell clones represents a clonal expansion of an epithelial stem cell present in the columnar epithelial tissue sample, thereby isolating epithelial stem cells.