Method of differentiating stem cells into cells of the endoderm and 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 differentiation in the direction of mesendoderm, wherein the process of stem cell differentiation is initiated by adding an effective amount of a bone morphogenetic protein ranging from 10 ng/ml to 50 ng/ml within the first four days of initiating the stem cell culture;
- (b) culturing the cells from step (a) under conditions favoring the formation of intact embryoid bodies (EBs), wherein the EBs are surrounded by a layer of visceral yolk sac; and
(c) culturing the cells from the EBs of step (b) under conditions favoring terminal differentiation of the cells to the pancreatic lineage.
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Abstract
Methods are described to more efficiently produce cells of the endoderm and pancreatic lineage from mammalian pluripotent stem cells. These methods provide a simple, reproducible culture protocol using defined media components to enable consistent, large-scale production of pancreatic cell types for research or therapeutic uses.
20 Citations
23 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
(a) culturing the stem cells under conditions that induce differentiation in the direction of mesendoderm, wherein the process of stem cell differentiation is initiated by adding an effective amount of a bone morphogenetic protein ranging from 10 ng/ml to 50 ng/ml within the first four days of initiating the stem cell culture; -
(b) culturing the cells from step (a) under conditions favoring the formation of intact embryoid bodies (EBs), wherein the EBs are surrounded by a layer of visceral yolk sac; and (c) culturing the cells from the EBs of step (b) under conditions favoring terminal differentiation of the cells to the pancreatic lineage. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 23)
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14. A method of sequentially enriching a culture derived from human pluripotent stem cells for cells of endoderm and pancreatic lineages, the method comprising the steps of
(a) culturing the stem cells under conditions that induce differentiation in the direction of mesendoderm, wherein the process of stem cell differentiation is initiated by adding an effective amount of a bone morphogenetic protein ranging from 10 ng/ml to 100 ng/ml and fibroblast growth factor ranging from 100 ng/ml to 200 ng/ml within the first four days of initiating the stem cell culture; -
(b) culturing the cells from step (a) under conditions favoring the formation of intact embryoid bodies (EBs), wherein the EBs are surrounded by a layer of visceral yolk sac; and (c) culturing the cells from the EBs of step (b) under conditions favoring terminal differentiation of the cells to the pancreatic lineage. - View Dependent Claims (15, 16, 17)
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18. A method of culturing human pluripotent stem cells to prepare a cell population of the pancreatic lineage, which does not have tumorigenic capability, the method comprising the steps of:
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(a) culturing the stem cells under conditions that induce differentiation in the direction of mesendoderm, wherein the process of stem cell differentiation is initiated by adding an effective amount of a bone morphogenetic protein ranging from 10 ng/ml to 50 ng/ml within the first four days of initiating the stem cell culture; (b) culturing the cells from step (a) under conditions favoring the formation of intact embryoid bodies (EBs), wherein the EBs are surrounded by a layer of visceral yolk sac; (c) culturing the cells from the EBs of step (b) under conditions favoring terminal differentiation of the cells to the pancreatic lineage; and (d) selecting for expression of a cell surface marker indicative of a commitment to a particular differentiated lineage, wherein the marker is EpCAM, the resulting cell culture not forming teratomas when injected in immunocompromised mice. - View Dependent Claims (19, 20, 21, 22)
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Specification