GENERATION OF INDUCED PLURIPOTENT STEM CELL LINES FROM HUMAN PATIENTS WITH MUTATIONS IN THE GLUCOKINASE GENE
Heterozygous and homozygous mutations in the glucokinase (GCK) gene lead to maturity-onset diabetes of the young type 2 (MODY2) and permanent neonatal diabetes (PNDM), respectively. The present invention relates to a method for generating induced pluripotent stem cell (iPSC) lines from patients with MODY2 and PNDM due to mutations in the GCK gene. The generated iPSC lines are able to differentiate into the three germ layers and show normal karyotypes. These iPSC lines can serve as valuable human cell models for understanding diabetes pathogenesis and developing new therapies for diabetes.
1 - 17 . (canceled)
18 . A method for generation of induced pluripotent stem cell (iPSC) lines from patients with mutations in a gene encoding glucokinase (GCK), the method comprising:
a) obtaining peripheral blood mononuclear cells (PBMCs) of patients with mutations in the GCK gene, wherein heterozygous mutations in the GCK gene cause maturity-onset diabetes of the young type 2 (MODY2), and homozygous mutations in the GCK gene cause permanent neonatal diabetes mellitus (PNDM);
b) identifying heterozygous or homozygous mutations in the GCK gene in the PBMCs using whole exome sequencing (WES);
c) confirming the heterozygous or homozygous mutations in the GCK gene in the PBMCs using Sanger sequencing;
d) reprogramming the PBMCs into the iPSC lines;
e) selecting and expanding the reprogrammed iPSC lines;
f) confirming the heterozygous or homozygous mutations in the GCK gene in the iPSC lines using Sanger sequencing; and
g) confirming the expression of pluripotency markers in the iPSC lines.
19 . The method according to claim 1 , further comprising using the iPSC lines to produce normal pancreatic beta-cells.
20 . The method according to claim 2 , wherein the normal pancreatic beta-cells are used in transplantation therapy.
21 . The method according to claim 1 , wherein the pluripotency markers comprise at least one of OCT4, NANOG, SOX2, TRA-1-60, TRA81, and SSEA4.
22 . The method according to claim 4 , wherein the pluripotency markers comprise OCT4.
23 . The method according to claim 4 , wherein the pluripotency markers comprise NANOG.
24 . The method according to claim 4 , wherein the pluripotency markers comprise SOX2.
25 . The method according to claim 4 , wherein the pluripotency markers comprise TRA-1-60.
26 . The method according to claim 4 , wherein the pluripotency markers comprise TRA81.
27 . The method according to claim 4 , wherein the pluripotency markers comprise SSEA4.
28 . The method according to claim 1 , wherein the iPSC lines form embryoid bodies (EBs) upon spontaneous differentiation and express specific markers of the three germ layers, including NESTIN and NEUROD1 (ectoderm), brachyury (T) (mesoderm), and SOX17 (endoderm).
29 . Induced pluripotent stem cells (iPSC) from patients with mutations in a gene encoding glucokinase (GCK), made by a method comprising:
a) obtaining peripheral blood mononuclear cells (PBMCs) of patients with mutations in the GCK gene, wherein heterozygous mutations in the GCK gene cause maturity-onset diabetes of the young type 2 (MODY2), and homozygous mutations in the GCK gene cause permanent neonatal diabetes mellitus (PNDM);
b) identifying heterozygous or homozygous mutations in the GCK gene in the PBMCs using whole exome sequencing (WES);
c) confirming the heterozygous or homozygous mutations in the GCK gene in the PBMCs using Sanger sequencing;
d) reprogramming the PBMCs into the iPSC lines;
e) selecting and expanding the reprogrammed iPSC lines;
f) confirming the heterozygous or homozygous mutations in the GCK gene in the iPSC lines using Sanger sequencing; and
g) confirming the expression of pluripotency markers in the iPSC lines.
30 . The induced pluripotent stem cells according to claim 12 , wherein the method further comprises using the iPSC lines to produce normal pancreatic beta-cells, and wherein the normal pancreatic beta-cells are used in transplantation therapy.
31 . The induced pluripotent stem cells according to claim 12 , wherein the pluripotency markers comprise at least one of OCT4, NANOG, SOX2, TRA-1-60, TRA81, and SSEA4.
32 . The induced pluripotent stem cells according to claim 14 , wherein the pluripotency markers comprise OCT4.
33 . The induced pluripotent stem cells according to claim 14 , wherein the pluripotency markers comprise NANOG.
34 . The induced pluripotent stem cells according to claim 14 , wherein the pluripotency markers comprise SOX2.
35 . The induced pluripotent stem cells according to claim 14 , wherein the pluripotency markers comprise TRA-1-60.
36 . The induced pluripotent stem cells according to claim 14 , wherein the pluripotency markers comprise TRA81.
37 . The induced pluripotent stem cells according to claim 14 , wherein the pluripotency markers comprise SSEA4.