Differentiation and Enrichment of Islet-Like Cells from Human Pluripotent Stem Cells
Methods for differentiating human pluripotent stem cells into islet-like cells are provided. In certain embodiments, the methods utilize sequential culturing of the human pluripotent stem cells with certain factors to produce islet-like cells. In certain embodiments, the population of cells produced by the methods is further enriched for islet-like cells.
1 . A method of enriching the proportion of islet-like cells from a cell population generated by the differentiation of human pluripotent stem cells to islet-like cells, comprising
a) differentiating human pluripotent stem cells into a cell population comprising islet-like cells, wherein the cell population comprises cell clusters that have formed buds; and
b) separating at least some of the buds from at least some of the clusters in the cell population into at least two fractions;
wherein at least one of the at least two resulting fractions contains a higher proportion of islet-like cells than the cell population.
2 . The method of claim 1 , wherein the islet-like cells comprise insulin-producing cells or glucagon-producing cells.
3 . The method of claim 1 , wherein the human pluripotent stem cells are human embryonic stem cells.
4 . The method of claim 1 , wherein the buds are separated from the clusters based on size differences between the buds and the clusters.
5 . The method of claim 4 , wherein the buds are separated from the clusters by use of nylon mesh.
6 . The method of claim 5 , wherein the nylon mesh is a 200 micron mesh.
7 . The method of claim 5 , wherein the nylon mesh is a 70 micron mesh.
8 . The method of claim 5 , wherein the nylon mesh is between 50 and 250 microns.
9 . The method of claim 4 , wherein at least one of the fractions with a higher proportion of islet-like cells than the cell population is a fraction with a smaller particle size than at least one of the other fractions that result from the size separation.
10 . The method of claim 9 , wherein the at least one fraction with a higher proportion of islet-like cells than the cell population has a particle size equal to or less than 200 microns.
11 . The method of claim 9 wherein the at least one fraction with a higher proportion of islet-like cells than the cell population has a particle size equal to or less than 150 microns.
12 . The method of claim 9 , wherein the at least one fraction with a higher proportion of islet-like cells than the cell population has a particle size equal to or less than 100 microns.
13 . The method of claim 4 , wherein the cell population is fractionated into three fractions by performing two size separation steps, wherein the second separation step is performed on a fraction that results from the first separation step.
14 . The method of claim 13 , wherein the first separation step is performed using a size separation value equal to or less than 50 microns and the second separation step is performed using a size separation value between 100 microns and 300 microns on the retained fraction from the first separation step; and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population.
15 . The method of claim 14 , wherein the first separation step is performed using a size separation value of 50 microns and the second separation step is performed using a size separation value of 150 microns, and wherein the 50 to 150 micron fraction contains higher proportion of islet-like cells than the cell population.
16 . The method of claim 13 , wherein the first separation step is performed using a size separation value equal to or less than 50 microns and the second separation step is performed using a size separation value between 150 microns and 250 microns on the retained fraction from the first separation step; and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population.
17 . The method of claim 16 , wherein the first separation step is performed using a size separation value of 50 microns and the second separation step is performed using a size separation value of 200 microns, and wherein the 50 to 200 micron fraction contains higher proportion of islet-like cells than the cell population.
18 . The method of claim 13 , wherein the first separation step is performed using a size separation value equal to or less than 20 microns and the second separation step is performed using a size separation value of 100 microns or greater on the retained fraction from the first separation step; and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population.
19 . The method of claim 18 , wherein the first separation step is performed using a size separation value of 20 microns and the second separation step is performed using a size separation value of 200 microns, and wherein the 20 to 200 micron fraction contains higher proportion of islet-like cells than the cell population.
20 . The method of claim 1 , wherein the buds are dissociated from the clusters before the separation step b).
21 . The method of claim 20 , wherein the buds are dissociated from the clusters by repeated pipetting.
22 . The method of claim 20 , wherein the buds are dissociated from the clusters by enzyme treatment.
23 . The method of claim 22 , wherein the enzyme is Dispase.
24 . The method of claim 22 , wherein the enzyme is trypsin.
25 . The method of claim 1 , wherein the buds are separated from the clusters by manually or robotically picking the buds.
26 . The method of claim 1 , wherein the buds are separated from the clusters based on density differences between the buds and the clusters.
27 . The method of claim 26 , wherein the buds are separated from at least some of the clusters using a Percoll gradient.
28 . The method of claim 4 , wherein the buds are separated from at least some of the clusters using flow cytometry.