AMNIOTIC FLUID DERIVED CELLS
This invention relates to an expandable population of amniotic fluid-derived cells that can be differentiated into a β-cell lineage. This invention also provides methods for isolating and expanding such amniotic fluid-derived cells, as well as related methods and compositions for utilizing such cells in the therapeutic treatment of diabetes.
1 . A substantially pure population of amniotic fluid-derived cells.
2 . The population of amniotic fluid-derived cells according to claim 1 , wherein the cells of said population are substantially negative in the expression of at least one protein marker selected from the group consisting of: CD117, Oct4, and Tra2-54.
3 . The population of amniotic fluid-derived cells according to claim 2 , wherein the cells of said population are substantially positive for the expression of at least one protein marker selected from the group consisting of: HNF-1 beta, GATA-6, and Sox-17.
4 . The population of amniotic fluid-derived cells according to claim 2 , wherein the cells of said population also express at least one gene selected from the group consisting of: HNF-3 beta, Hes-1, GATA-4 and Musashi-1.
5 . The population of amniotic fluid-derived cells according to claim 2 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, AXL, bFGF, EGF receptor, Fas/TNFRSF6, GRO, GRO-alpha, ICAM-1, IL-1 alpha, Il-3, Il-6, Il-8, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
6 . The population of amniotic fluid-derived cells according to claim 2 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17.
7 . The population of amniotic fluid-derived cells according to claim 6 , wherein the cells of said population are substantially negative in the expression of cytokeratin protein.
8 . The population of amniotic fluid-derived cells according to claim 6 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, bFGF, EGF receptor, GRO, GRO-alpha, ICAM-1, Il-3, Il-6, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
9 . The population of amniotic fluid-derived cells according to claim 1 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17, and also express at least one gene selected from the group consisting of: Hes-1 and Musashi-1.
10 . The population of amniotic fluid-derived cells according to claim 1 , wherein the cells of said population are substantially negative in the expression of CD 117, Oct4, Sox-17, and Tra2-54.
11 . The population of amniotic fluid-derived cells according to claim 10 , wherein the cells of said population are substantially negative in the expression of cytokeratin protein.
12 . The population of amniotic fluid-derived cells according to claim 10 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, bFGF, EGF receptor, GRO, GRO-alpha, ICAM-1, Il-3, Il-6, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
13 . The population of amniotic fluid-derived cells according to claim 10 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17, and also express at least one gene selected from the group consisting of: Hes-1 and Musashi-1.
14 . The population of pancreatic amniotic fluid-derived cells according to claim 1 , capable of propagating in vitro.
15 . The population of amniotic fluid-derived cells according to claim 1 , capable of propagating in vitro under hypoxic conditions.
16 . The population of amniotic fluid-derived cells according to claim 1 , capable of differentiating into cells displaying the characteristics of the β-cell lineage.
17 . The population of amniotic fluid-derived cells according to claim 1 , capable of differentiating into a gut hormone-producing cell.
18 . A method of obtaining a population of cells from amniotic fluid, comprising:
a. Isolating amniotic fluid,
b. Isolating the cells from the amniotic fluid,
c. Placing the cells in culture medium,
d. Plating the cells in a culture vessel, and
e. Allowing the cells to grow in said medium for at least about five days, thereby obtaining a population of amniotic fluid-derived cells.
19 . The method according to claim 18 , wherein the cells of said population are substantially negative in the expression of at least one protein marker selected from the group consisting of: CD117, Oct4, and Tra2-54.
20 . The method according to claim 19 , wherein the cells of said population are substantially positive for the expression of at least one protein marker selected from the group consisting of: HNF-1 beta, GATA-6, and Sox-17.
21 . The method according to claim 19 , wherein the cells of said population also express at least one gene selected from the group consisting of: HNF-3 beta, Hes-1, GATA-4 and Musashi-1.
22 . The method according to claim 19 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, AXL, bFGF, EGF receptor, Fas/TNFRSF6, GRO, GRO-alpha, ICAM-1, IL-1 alpha, Il-3, Il-6, Il-8, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
23 . The method according to claim 19 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17.
24 . The method according to claim 23 , wherein the cells of said population are substantially negative in the expression of cytokeratin protein.
25 . The method according to claim 23 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, bFGF, EGF receptor, GRO, GRO-alpha, ICAM-1, Il-3, Il-6, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
26 . The method according to claim 18 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17, and also express at least one gene selected from the group consisting of: Hes-1 and Musashi-1.
27 . The method according to claim 18 , wherein the cells of said population are substantially negative in the expression of CD117, Oct4, Sox-17, and Tra2-54.
28 . The method according to claim 27 , wherein the cells of said population are substantially negative in the expression of cytokeratin protein.
29 . The method according to claim 27 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, bFGF, EGF receptor, GRO, GRO-alpha, ICAM-1, Il-3, Il-6, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
30 . The method according to claim 27 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17, and also express at least one gene selected from the group consisting of: Hes-1 and Musashi-1.
31 . The method according to claim 18 , wherein the cells of said population are capable of propagating in vitro.
32 . The method according to claim 18 , wherein the cells of said population are capable of propagating in vitro under hypoxic conditions.
33 . The method according to claim 18 , wherein the cells of said population are capable of differentiating into cells displaying the characteristics of the β-cell lineage.
34 . The method according to claim 18 , wherein the cells of said population are capable of differentiating into a gut hormone-producing cell.
35 . A method of obtaining a population of cells from amniotic fluid, comprising:
a. Isolating amniotic fluid,
b. Isolating the cells from the amniotic fluid,
c. Selecting cells that express at least one of the markers selected from the group consisting of SSEA-4, SSEA-3, TRA1-60 and TRA1-81,
d. Placing the cells in culture medium,
e. Plating the cells in a culture vessel, and
f. Allowing the cells to grow in said medium for at least about five days, thereby obtaining a population of amniotic fluid-derived cells.
36 . The method according to claim 35 , wherein the cells of said population are substantially negative in the expression of at least one protein marker selected from the group consisting of: CD117, Oct4, and Tra2-54.
37 . The method according to claim 36 , wherein the cells of said population are substantially positive for the expression of at least one protein marker selected from the group consisting of: HNF-1 beta, GATA-6, and Sox-17.
38 . The method according to claim 36 , wherein the cells of said population also express at least one gene selected from the group consisting of: HNF-3 beta, Hes-1, GATA-4 and Musashi-1.
39 . The method according to claim 36 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, AXL, bFGF, EGF receptor, Fas/TNFRSF6, GRO, GRO-alpha, ICAM-1, IL-1 alpha, Il-3, Il-6, Il-8, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
40 . The method according to claim 36 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17.
41 . The method according to claim 40 , wherein the cells of said population are substantially negative in the expression of cytokeratin protein.
42 . The method according to claim 40 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, bFGF, EGF receptor, GRO, GRO-alpha, ICAM-1, Il-3, Il-6, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
43 . The method according to claim 35 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17, and also express at least one gene selected from the group consisting of: Hes-1 and Musashi-1.
44 . The method according to claim 40 , wherein the cells of said population are substantially negative in the expression of CD117, Oct4, Sox-17, and Tra2-54.
45 . The method according to claim 44 , wherein the cells of said population are substantially negative in the expression of cytokeratin protein.
46 . The method according to claim 44 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, bFGF, EGF receptor, GRO, GRO-alpha, ICAM-1, Il-3, Il-6, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
47 . The method according to claim 44 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17, and also express at least one gene selected from the group consisting of: Hes-1 and Musashi-1.
48 . The method according to claim 35 , wherein the cells of said population are capable of propagating in vitro.
49 . The method according to claim 35 , wherein the cells of said population are capable of propagating in vitro under hypoxic conditions.
50 . The method according to claim 35 , wherein the cells of said population are capable of differentiating into cells displaying the characteristics of the β-cell lineage.
51 . The method according to claim 35 , wherein the cells of said population are capable of differentiating into a gut hormone-producing cell.
52 . A method of treating a patient with diabetes mellitus or at risk of developing diabetes, comprising:
a. Isolating a population of amniotic fluid-derived cells from a donor, and
b. Transferring the cells into the patient.
53 . The method according to claim 52 , wherein the cells of said population are substantially negative in the expression of at least one protein marker selected from the group consisting of: CD117, Oct4, and Tra2-54.
54 . The method according to claim 53 , wherein the cells of said population are substantially positive for the expression of at least one protein marker selected from the group consisting of: HNF-1 beta, GATA-6, and Sox-17.
55 . The method according to claim 53 , wherein the cells of said population also express at least one gene selected from the group consisting of: HNF-3 beta, Hes-1, GATA-4 and Musashi-1.
56 . The method according to claim 53 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, AXL, bFGF, EGF receptor, Fas/TNFRSF 6 , GRO, GRO-alpha, ICAM-1, IL-1 alpha, Il-3, Il-6, Il-8, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
57 . The method according to claim 53 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17.
58 . The method according to claim 57 , wherein the cells of said population are substantially negative in the expression of cytokeratin protein.
59 . The method according to claim 57 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, bFGF, EGF receptor, GRO, GRO-alpha, ICAM-1, Il-3, Il-6, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
60 . The method according to claim 52 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17, and also express at least one gene selected from the group consisting of: Hes-1 and Musashi-1.
61 . The method according to claim 57 , wherein the cells of said population are substantially negative in the expression of CD117, Oct4, Sox-17, and Tra2-54.
62 . The method according to claim 61 , wherein the cells of said population are substantially negative in the expression of cytokeratin protein.
63 . The method according to claim 61 , wherein the cells of said population are positive for the expression of at least one protein marker selected from the group consisting of: Liver Activator Protein, PDGF receptor β, bFGF, EGF receptor, GRO, GRO-alpha, ICAM-1, Il-3, Il-6, MIF, Osteoprotegerin, TIMP-2, and TRAIL R3.
64 . The method according to claim 61 , wherein the cells of said population are substantially negative in the expression of the protein marker Sox-17, and also express at least one gene selected from the group consisting of: Hes-1 and Musashi-1.
65 . The method according to claim 52 , wherein the cells of said population are differentiated into pancreatic hormone producing cells prior to the step of transferring into the patient.
66 . The method according to claim 52 , wherein the cells of said population differentiate into pancreatic hormone producing cells after the step of transferring into the patient.
67 . The method according to claim 52 , wherein the cells of said population are capable of propagating in vitro.
68 . The method according to claim 52 , wherein the cells of said population are capable of propagating in vitro under hypoxic conditions.
69 . The method according to claim 52 , wherein the cells of said population are capable of differentiating into cells displaying the characteristics of the β-cell lineage.
70 . The method according to claim 52 , wherein the cells of said population are capable of differentiating into a gut hormone-producing cell.