Derivation of human induced pluripotent stem cells from patients with maturity onset diabetes of the young

Abstract

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human "stem cell cassette" containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1-60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.

FIGURE 1.

Summary of individuals recruited for this study. A, family node pedigrees for MODY1, MODY2, MODY3, MODY5, and MODY8 families. Squares denote males, circles denote females, solid symbols denote diabetes, NN denotes no mutation and NM denotes mutation. B, clinical characteristics of individuals in the study. Individuals were de-identified and assigned an alternate identification number (ID). Age (years old) at which skin biopsies and skin fibroblasts were obtained, gender (male or female), and source of fibroblasts are indicated. Type of MODY condition (MODY1, MODY2, MODY3, MODY5, or MODY8), specific disease variant in the MODY gene, presence or absence of mutation (yes or no), and presence or absence of diabetes (yes or no) are indicated.

FIGURE 2.

Derivation of hiPSCs from healthy individuals and MODY patients. A–E, MODY gene mutations were confirmed in MODY1 (p.Ile271fs) (N904-2 and N904-6) (A), MODY2 (V62A) (N2-6, N2-9, and N2-10) (B), MODY3 (P291fsinsC) (N26-3 and N26-7) (C), MODY5 (S148L) (N805-1 and N805-2) (D), and MODY8 (C563fsX673) (N65-102) (E) patient fibroblasts. F, the hiPSCs derived in this study morphologically resemble hESCs (CHB8, Daley laboratory). Scale bar: 200 μm. G, hiPSCs express endogenous OCT4 and NANOG transcripts at comparable (high) levels to a control hESC line (CHB8). H, immunostaining analyses for OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60 in a representative hiPSC line (MODY5; iN805-2). Scale bar: 200 μm. I, hiPSCs give rise to derivatives of the three germ layers: ectoderm (primitive brain), mesoderm (cartilage), and definitive endoderm (respiratory epithelium) in an in vivo teratoma assay. Scale bar: 200 μm. J, hiPSCs exhibit a normal karyotype in a G-band analysis.