RE-AGGREGATION OF STEM CELL-DERIVED PANCREATIC BETA CELLS

1 . An in vitro cell cluster comprising at least one non-native pancreatic β cell that exhibits an in vitro glucose-stimulated insulin secretion response when exposed to a glucose challenge, wherein the cell cluster is an unsorted cell cluster, and wherein the cell cluster comprises:

(a) at least about 35% cells that express NKX6.1 and C-peptide;

(b) at least about 70% cells that express chromogranin A;

(c) at most about 2% cells that express SOX2; and

(d) at most about 10% cells that express SOX9, as measured by flow cytometry.

2 - 27 . (canceled)

28 . A composition that comprises an in vitro cell cluster suspended in a serum-free culture medium, wherein the in vitro cell cluster comprises:

(i) at least about 35% cells that express NKX6.1 and C-peptide;

(ii) at least about 70% cells that express chromogranin A,

(iii) at most about 2% cells that express SOX2, and

(iv) at most about 10% cells that express SOX9, as measured by flow cytometry.

29 - 43 . (canceled)

44 . A method, comprising:

(a) obtaining a first cell cluster comprising at least one NKX6.1-positive and C-peptide-positive cell;

(b) dissociating a plurality of cells from the first cell cluster; and

(c) culturing the plurality of cells from (b) in a serum-free culture medium, thereby:

(1) allowing at least a portion of the plurality of cells to form a second in vitro cell cluster, and

(2) differentiating a portion of cells in the second cell cluster into non-native β cells in vitro, wherein said non-native pancreatic β cells exhibit an in vitro glucose-stimulated insulin secretion response when exposed to a glucose challenge, and wherein secretion of insulin by the non-native pancreatic β cells in response to a glucose challenge is proportional to glucose concentration in the glucose challenge.

45 . (canceled)

46 . The method of claim 44 ,

wherein:

(i) the second in vitro cell cluster comprises a higher percentage cells that express chromogranin A as compared the first cell cluster;

(ii) the second in vitro cell cluster comprises a higher percentage cells that express NKX6.1 and C-peptide as compared the first cell cluster;

(iii) the second in vitro cell cluster comprises a lower percentage cells that express SOX2 as compared the first cell cluster; or

(iv) the second in vitro cell cluster comprises a lower percentage of cells that express SOX9 as compared the first cell cluster.

47 . The method of claim 44 ,

wherein the second in vitro cell cluster comprises:

(i) at least about 35% cells that express NKX6.1 and C-peptide;

(ii) at least about 70% cells that express chromogranin A;

(iii) at most about 2% cells that express SOX2; or

(iv) at most about 10% cells that express SOX9.

48 . (canceled)

49 . The method of claim 44 , wherein the dissociating comprises enzymatic dissociation of the first cell cluster.

50 . (canceled)

51 . (canceled)

52 . The method of claim 44 , wherein the culture medium is stirred at a predetermined speed.

53 . The method of claim 52 , wherein the predetermined speed is from about 30 rpm to about 60 rpm.

54 - 58 . (canceled)

59 . The method of claim 44 , wherein the non-native pancreatic β cells exhibit an in vitro glucose-stimulated insulin secretion response to a first glucose challenge, a second glucose challenge, and a third glucose challenge, when the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

60 . The method of claim 44 , wherein the non-native pancreatic β cells are genetically modified.

61 . The method of claim 44 , wherein secretion of insulin by the non-native pancreatic β cells in response to a glucose challenge is proportional to glucose concentration in the glucose challenge.

62 - 65 . (canceled)

66 . The method of claim 44 , wherein the serum-free culture medium does not comprise an exogenous inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK).

67 . The method of claim 44 , wherein the serum-free culture medium does not comprise an exogenous extracellular matrix molecule.

68 . The method of claim 44 , wherein the first cell cluster is recovered from cryopreservation.

69 - 71 . (canceled)

72 . The method of claim 44 , wherein the second in vitro cell cluster has a diameter that is at most 50% of the first cell cluster.

73 - 79 . (canceled)

80 . The method of claim 44 , wherein the second in vitro cell cluster comprises at least about 40% cells that express both NKX6.1 and C-peptide.

81 . The method of claim 44 , wherein a percentage of cells in the second in vitro cell cluster that express SOX2 is at least 5 times lower than a percentage of cells in the first cell cluster that express SOX2.

82 . (canceled)

83 . The method of claim 44 , wherein a percentage of cells in the second in vitro cell cluster that express SOX9 is at least 5 times lower than a percentage of cells in the first cell cluster that express SOX9.

84 - 88 . (canceled)

89 . The method of claim 44 , wherein the second in vitro cell cluster exhibits a higher stimulation index compared to the first cell cluster, wherein the stimulation index equals to a ratio of insulin secreted in response to a first glucose concentration as compared to a second glucose concentration, and wherein the first glucose concentration is higher than the second glucose concentration.

90 - 97 . (canceled)

98 . An in vitro cell cluster that is generated by the method of claim 44 .

99 . A device that comprises an in vitro cell cluster or a portion thereof, wherein the in vitro the cell cluster comprises:

(a) at least about 35% cells that express NKX6.1 and C-peptide;

(b) at least about 70% cells that express chromogranin A;

(c) at most about 2% cells that express SOX2; and

(d) at most about 10% cells that express SOX9, as measured by flow cytometry, and

wherein the device is configured to produce and release insulin when implanted into a subject.

100 - 105 . (canceled)

106 . A method of treating or preventing a disease in a subject in need thereof, the method comprising administering a cell cluster or a portion thereof to the subject, wherein the in vitro cell cluster comprises:

(a) at least about 35% cells that express NKX6.1 and C-peptide;

(b) at least about 70% cells that express chromogranin A;

(c) at most about 2% cells that express SOX2; and

(d) at most about 10% cells that express SOX9, as measured by flow cytometry.

107 . A method of treating or preventing a disease in a subject in need thereof, the method comprising implanting a device to the subject, wherein the device comprises an in vitro cell cluster, wherein the in vitro cell cluster comprises:

(a) at least about 35% cells that express NKX6.1 and C-peptide;

(b) at least about 70% cells that express chromogranin A;

(c) at most about 2% cells that express SOX2; and

(d) at most about 10% cells that express SOX9, as measured by flow cytometry.

108 - 163 . (canceled)

164 . A pharmaceutical composition that comprises an in vitro cell cluster or a portion thereof and at least one pharmaceutically acceptable excipient, wherein the in vitro cell cluster comprises:

(a) at least about 35% cells that express NKX6.1 and C-peptide;

(b) at least about 70% cells that express chromogranin A;

(c) at most about 2% cells that express SOX2; and

(d) at most about 10% cells that express SOX9, as measured by flow cytometry.

165 - 169 . (canceled)

170 . A kit comprising (i) a cell cluster comprising at least one non-native pancreatic β cell, wherein at least about 70% of cells in the cell cluster express chromogranin A as measured by flow cytometry, and wherein the cell cluster exhibits an in vitro glucose-stimulated insulin secretion response when exposed to a glucose challenge, and (ii) a buffer.

171 . (canceled)

172 . A cell composition that comprises:

(a) at least about 30% cells expressing PDX 1 and NKX6.1;

(b) at least about 20% cells expressing C-peptide and NKX6.1;

(c) at least about 40% cells expressing CHGA;

(d) at most about 30% cells expressing Cdx2; and

(e) at most about 10% cells expressing SOX2, as measured by flow cytometry,

wherein said cell composition is a reconstituted cryopreserved cell composition.