Markers for differentiation of stem cells into differentiated cell populations
First Claim
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1. A method of identifying differentiation markers expressed in pancreatic β
- -cell lineage cells, comprising;
(a) culturing an isolated primary cell under conditions that comprise exogenously expressing Oct4, Sox2, cMyc, and Klf4 in said primary cell, such that said primary cell de-differentiates to generate an induced pluripotent stem cell (iPSC);
(b) treating said iPSC under conditions such that said iPSC differentiates into a pancreatic β
-cell, wherein said treating comprises;
(i) culturing the iPSC in a chemically defined medium comprising fibroblast growth factor, Activin A and bone morphogenetic protein,(ii) culturing the cells produced from step (i) in the presence of chemically defined medium comprising insulin, transferrin, selenium, a fibroblast growth factor and nicotinamide,(iii) culturing the cells produced from step (ii) in the presence of a chemically defined medium comprising insulin, transferrin, selenium, retinoic acid, a bone morphogenetic protein inhibitor and nicotinamide;
and (iv) culturing the cells produced from step (iii) in the presence of a serum-free medium comprising an insulin-like growth factor, insulin, nicotinamide, exendin-4 and/or GLP-1, a TGF-β
inhibitor, and an agent that increases cAMP; and
(c) identifying molecular markers expressed during differentiation, wherein said markers are indicative of differentiation of said iPS cell into a pancreatic β
-cell.
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Abstract
Provided herein are systems and methods for identifying cell-specific differentiation markers. In particular, provided herein are systems and methods for generating induced pluripotent cells (IPS) from human cells, differentiating the IPS into differentiated cells, and identifying differentiation specific markers.
23 Citations
15 Claims
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1. A method of identifying differentiation markers expressed in pancreatic β
- -cell lineage cells, comprising;
(a) culturing an isolated primary cell under conditions that comprise exogenously expressing Oct4, Sox2, cMyc, and Klf4 in said primary cell, such that said primary cell de-differentiates to generate an induced pluripotent stem cell (iPSC); (b) treating said iPSC under conditions such that said iPSC differentiates into a pancreatic β
-cell, wherein said treating comprises;(i) culturing the iPSC in a chemically defined medium comprising fibroblast growth factor, Activin A and bone morphogenetic protein, (ii) culturing the cells produced from step (i) in the presence of chemically defined medium comprising insulin, transferrin, selenium, a fibroblast growth factor and nicotinamide, (iii) culturing the cells produced from step (ii) in the presence of a chemically defined medium comprising insulin, transferrin, selenium, retinoic acid, a bone morphogenetic protein inhibitor and nicotinamide; and (iv) culturing the cells produced from step (iii) in the presence of a serum-free medium comprising an insulin-like growth factor, insulin, nicotinamide, exendin-4 and/or GLP-1, a TGF-β
inhibitor, and an agent that increases cAMP; and(c) identifying molecular markers expressed during differentiation, wherein said markers are indicative of differentiation of said iPS cell into a pancreatic β
-cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
- -cell lineage cells, comprising;
Specification