METHODS AND DEVICES FOR DIFFERENTIATING PLURIPOTENT STEM CELLS INTO CELLS OF THE PANCREATIC LINEAGE
First Claim
1. A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:
- (a) culturing the stem cells under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells in a chemically defined medium comprising an effective amount ofi) fibroblast growth factor,ii) Activin A, andiii) bone morphogenetic protein;
(b) culturing the cells from step (a) in the presence of a chemically defined medium comprising an effective amount of insulin, transferrin, and selenium, wherein the medium further comprises a fibroblast growth factor in an amount that ranges from about 10 ng/ml to about 200 ng/ml; and
(c) culturing the cells under conditions to produce foregut/pancreatic progenitor PDX1+ Ins−
cells.
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Abstract
Methods and devices for culturing human pluripotent stem cells to produce cells of the pancreatic lineage are disclosed. The methods include steps of culturing the stem cells under conditions that induce the expression of mesendoderm/primitive streak and definitive endoderm markers in a chemically defined medium including an effective amount of i) fibroblast growth factor, ii) Activin A, and iii) bone morphogenetic protein. The methods further include the steps of culturing cells under conditions favoring the formation of at least one of intact embryoid bodies and pancreatic progenitor PDX1+ Ins− cells.
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Citations
28 Claims
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1. A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:
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(a) culturing the stem cells under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells in a chemically defined medium comprising an effective amount of i) fibroblast growth factor, ii) Activin A, and iii) bone morphogenetic protein; (b) culturing the cells from step (a) in the presence of a chemically defined medium comprising an effective amount of insulin, transferrin, and selenium, wherein the medium further comprises a fibroblast growth factor in an amount that ranges from about 10 ng/ml to about 200 ng/ml; and (c) culturing the cells under conditions to produce foregut/pancreatic progenitor PDX1+ Ins−
cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:
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(a) culturing the stem cells under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells in a chemically defined medium comprising an effective amount of i) fibroblast growth factor, ii) Activin A, and iii) bone morphogenetic protein; (b) culturing the cells from step (a) under conditions favoring the formation of embryoid bodies; and (c) culturing the embryoid bodies under conditions favoring the formation of pancreas-spheres co-expressing PDX1, HNF1β
, HNF6, and Sox9 proteins. - View Dependent Claims (16, 17, 18, 19, 20, 21)
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22. A method of culturing pluripotent stem cells in a committed partially differentiated state, comprising the steps of:
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(a) culturing the stem cells under conditions that induce formation of embryoid bodies containing multipotent progenitor cells; and (b) culturing the embryoid bodies on an extracellular matrix in cell culture inserts having a porous floor so as to maintain the multipotent progenitors cells in a non-terminally differentiated state. - View Dependent Claims (23)
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24. A method of producing progenitor cells of the pancreatic lineage, comprising the steps of:
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(a) seeding human pluripotent stem cells in a cell culture vessel comprising an upper chamber and a lower chamber, wherein a bottom surface of the upper chamber comprises a porous substrate; (b) culturing the cells in a chemically defined medium comprising an effective amount of i) fibroblast growth factor, ii) Activin A, and iii) bone morphogenetic protein; and (c) obtaining pancreatic progenitor PDX1+ Ins−
cells. - View Dependent Claims (25, 26, 27)
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28. A pancreatic progenitor cell culture implant platform, comprising:
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(a) a bicameral cell culture system with an upper chamber and a lower chamber separated by a porous substrate; and (b) stem cells cultured in the upper chamber on the porous substrate under conditions that induce commitment to pancreatic progenitor PDX1+ Ins−
cells,wherein the stems cells may be treated in a single step to differentiate into insulin producing cells of the pancreatic lineage and either removed from the platform for implantation into a subject or implanted into a subject along with the porous substrate.
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Specification