US 11,739,366 B2
Methods for detection of rare subpopulations of cells and highly purified compositions of cells
Irina V. Klimanskaya, Upton, MA (US); and Roger Gay, Acton, MA (US)
Assigned to Astellas Institute for Regenerative Medicine, Westborough, MA (US)
Appl. No. 13/811,708
Filed by Irina V. Klimanskaya, Upton, MA (US); and Roger Gay, Acton, MA (US)
PCT Filed Jul. 25, 2011, PCT No. PCT/US2011/045232
§ 371(c)(1), (2), (4) Date Jul. 26, 2013,
PCT Pub. No. WO2012/012803, PCT Pub. Date Jan. 26, 2012.
Claims priority of provisional application 61/414,770, filed on Nov. 17, 2010.
Claims priority of provisional application 61/367,038, filed on Jul. 23, 2010.
Prior Publication US 2013/0302824 A1, Nov. 14, 2013
Int. Cl. G01N 33/53 (2006.01); C12Q 1/42 (2006.01); G01N 33/569 (2006.01); G01N 33/50 (2006.01)
CPC C12Q 1/42 (2013.01) [G01N 33/5005 (2013.01); G01N 33/56966 (2013.01)] 22 Claims
 
1. A method of confirming absence of contaminating pluripotent stem cells in a preparation of differentiated cells generated by differentiation of said pluripotent stem cells, comprising:
(a) providing a preparation of differentiated cells produced by in vitro differentiation of pluripotent stem cells in a differentiation medium, and culturing said preparation of differentiated cells in a stem cell medium, wherein the stem cell medium maintains pluripotent stem cells in their pluripotent state;
(b) applying a first stain and a second stain to said preparation of differentiated cells, wherein
said first stain detects a first embryonic stem cell marker, expression of which is indicative of presence of said contaminating pluripotent stem cells, and
said second stain detects a second embryonic stem cell marker, expression of which is indicative of presence of said contaminating pluripotent stem cells,
wherein said first embryonic stem cell marker is different from said second embryonic stem cell marker,
wherein said first embryonic stem cell maker is alkaline phosphatase; and
(c) confirming absence of contaminating pluripotent stem cells, if no cells positive for both said first stain and said second stain are identified by microscopic observation.