Differentiation of pluripotent stem cells
First Claim
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1. A method to derive a population of pancreatic endocrine precursor cells from pluripotent stem cells comprising the steps of:
- a. Culturing a population of pluripotent stem cells;
b. Differentiating the population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage;
c. Differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into a population of primitive gut tube cells;
d. Differentiating the population of primitive gut tube cells into a population of posterior foregut cells; and
e. Treating the population of posterior foregut cells with a medium supplemented with a CYP26A inhibitor and with no added retinoic acid such that the population of posterior foregut cells differentiates into a population of pancreatic endocrine precursor cells, wherein expression of endocrine precursor markers is increased and wherein the differentiating in steps b., c. and d. comprises treatment in a medium lacking a CPY26A inhibitor.
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Abstract
The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method utilizing a CYP26A inhibitor to produce a population of pancreatic endocrine precursor cells.
147 Citations
22 Claims
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1. A method to derive a population of pancreatic endocrine precursor cells from pluripotent stem cells comprising the steps of:
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a. Culturing a population of pluripotent stem cells; b. Differentiating the population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage; c. Differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into a population of primitive gut tube cells; d. Differentiating the population of primitive gut tube cells into a population of posterior foregut cells; and e. Treating the population of posterior foregut cells with a medium supplemented with a CYP26A inhibitor and with no added retinoic acid such that the population of posterior foregut cells differentiates into a population of pancreatic endocrine precursor cells, wherein expression of endocrine precursor markers is increased and wherein the differentiating in steps b., c. and d. comprises treatment in a medium lacking a CPY26A inhibitor. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method to derive a population of pancreatic endocrine precursor cells from pluripotent stem cells comprising the steps of:
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a. Differentiating pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage (Stage I) by the culturing the pluripotent stem cells in a medium supplemented with activin A; b. Differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage (Stage I) into a population of primitive gut tube cells (Stage II) by culturing the Stage I cells in a medium supplemented with FGF7; c. Differentiating the population of primitive gut tube cells (Stage II) into a population of posterior foregut cells (Stage III) by culturing the Stage II cells in a medium supplemented with retinoic acid and a P38 inhibitor; and d. Differentiating the population of posterior foregut cells (Stage III) into a population of Stage IV cells by treating the Stage III cells with a media supplemented with a CPY26A inhibitor and with no added retinoic acid, wherein the treatment increases the expression of endocrine precursor markers and wherein the differentiating in steps a., b. and c. comprises treatment in a medium lacking a CPY26A inhibitor. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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