iPSC-derived β cells model diabetes due to glucokinase deficiency
Haiqing Hua, Linshan Shang, Hector Martinez, Matthew Freeby, Mary Pat Gallagher, Thomas Ludwig, Liyong Deng, Ellen Greenberg, Charles LeDuc, Wendy K. Chung, Robin Goland, Rudolph L. Leibel, Dieter Egli
Haiqing Hua, Linshan Shang, Hector Martinez, Matthew Freeby, Mary Pat Gallagher, Thomas Ludwig, Liyong Deng, Ellen Greenberg, Charles LeDuc, Wendy K. Chung, Robin Goland, Rudolph L. Leibel, Dieter Egli
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iPSC-derived β cells model diabetes due to glucokinase deficiency

Abstract

Diabetes is a disorder characterized by loss of β cell mass and/or β cell function, leading to deficiency of insulin relative to metabolic need. To determine whether stem cell–derived β cells recapitulate molecular-physiological phenotypes of a diabetic subject, we generated induced pluripotent stem cells (iPSCs) from subjects with maturity-onset diabetes of the young type 2 (MODY2), which is characterized by heterozygous loss of function of the gene encoding glucokinase (GCK). These stem cells differentiated into β cells with efficiency comparable to that of controls and expressed markers of mature β cells, including urocortin-3 and zinc transporter 8, upon transplantation into mice. While insulin secretion in response to arginine or other secretagogues was identical to that in cells from healthy controls, GCK mutant β cells required higher glucose levels to stimulate insulin secretion. Importantly, this glucose-specific phenotype was fully reverted upon gene sequence correction by homologous recombination. Our results demonstrate that iPSC-derived β cells reflect β cell–autonomous phenotypes of MODY2 subjects, providing a platform for mechanistic analysis of specific genotypes on β cell function.

Authors

Haiqing Hua, Linshan Shang, Hector Martinez, Matthew Freeby, Mary Pat Gallagher, Thomas Ludwig, Liyong Deng, Ellen Greenberg, Charles LeDuc, Wendy K. Chung, Robin Goland, Rudolph L. Leibel, Dieter Egli

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Figure 1

An allelic series of GCK mutations in cells from a MODY2 subject.

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An allelic series of GCK mutations in cells from a MODY2 subject. (A) St...
(A) Structure of the GCK gene and nucleotide sequences of the mutations. Black boxes represent exons. The asterisks indicate the mutations (E256K and G299R). (B) Schematic view of the first step of the gene correction procedure: exons 7–10 of GCK, either the mutant or the wild-type allele, were replaced with a hygro-TK cassette. Sequences at the mutation site were analyzed by Sanger sequencing. P1 and P2 (blue arrows) were the primers used to detect integration of the hygro-TK (see Supplemental Table 2 for primer sequences). (C) Scheme of the second round of gene targeting replacing the hygro-TK cassette with the wild-type locus marked by an intronic SNP (triangle). Both targeting steps were facilitated by site-specific endonucleases, a ZFN for the first step and I-SceI for the second step. Green bars indicate the restriction sites, and the red bar indicates the probe used for Southern blot analysis. Blue bars represent primers used to screen and identify targeting events (see Supplemental Table 2 for primer sequences). PCR (with P1 and P3) and Sanger sequencing showed the corrected sequence at the mutation site and the intronic SNP that marks the corrected allele. (D) Southern blot analysis showing 2 bands representing the targeted allele (GCK+/hygro, 1.5 kb) and the nontargeted allele (GCK+/hygro or GCKG299R/hygro, 2.4 kb). (E) Karyotype analysis of GCKcorrected/+ cells.