title: Integration-Free Human Induced Pluripotent Stem Cells From Type 1 Diabetes Patient Skin Fibroblasts Show Increased Abundance of Pancreas-Specific microRNAs.
abstract: Type 1 diabetes (T1D) is a disease that is typically associated with multigenetic changes as well as environmental triggers. Disease-specific induced pluripotent stem cells (iPSCs) are preferable cell sources to study T1D, as they are derived from patient cells and therefore capture the disease genotype in a stem cell line. The purpose of this study was to generate integration-free iPSCs from adult skin fibroblasts with T1D. iPSCs were generated by transfection of synthetic mRNAs encoding transcription factors OCT4, SOX2, KLF4, c-MYC, and LIN28. Phase-contrast microscopy, immunocytochemistry, karyotyping, bisulfite genomic sequencing, reverse transcription-polymerase chain reaction, and teratoma formation assay were used to determine reprogramming efficiency, pluripotency, and differentiation potential. Following 18 consecutive days of synthetic mRNA transfections, the T1D patient skin fibroblasts underwent morphological changes, and the aggregated clumps exhibited a human embryonic stem cell (ESC)-like morphology with a high nucleus/cytoplasm ratio. Highly efficient generation of iPSCs was achieved using the mRNA reprogramming approach. The disease-specific iPSCs expressed pluripotency markers, maintained a normal karyotype, and formed teratomas containing tissues representative of the three germ layers when injected into immune-deficient mice. Of interest, the iPSCs showed upregulations of pancreas-specific microRNAs, compared with parental fibroblasts. These data indicate that T1D patient skin fibroblasts can be reprogrammed to pluripotency using a synthetic mRNA approach. These cells can serve as a useful tool for the identification of genes that are involved in autoimmune reactions as well as generating patient-matched β-cells for cell-based therapy.
authors: Cell Reprogramming and Stem Cells, Monash Institute of Medical Research, Monash University , Clayton, VIC , Australia.
authors: Jun
authors: J
authors: Liu
authors: † Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, The University of Sydney , Camperdown, NSW , Australia.
authors: Mugdha V
authors: MV
authors: Joglekar
authors: Cell Reprogramming and Stem Cells, Monash Institute of Medical Research, Monash University , Clayton, VIC , Australia.
authors: Huseyin
authors: H
authors: Sumer
authors: † Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, The University of Sydney , Camperdown, NSW , Australia.
authors: Anandwardhan A
authors: AA
authors: Hardikar
authors: ‡School of Public Health and Preventive Medicine, Monash University, Clayton, VIC, Australia; §Diabetes Unit, Southern Health Clayton, Clayton, VIC, Australia.
authors: Halena
authors: H
authors: Teede
authors: Cell Reprogramming and Stem Cells, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia; ¶Turretfield Research Centre, South Australian Research and Development Institute, Rosedale SA 3050, Australia.
authors: Paul J
authors: PJ
authors: Verma
keywords: Induced pluripotent stem cells (iPSCs); Pluripotency reprogramming; Type 1 diabetes (T1D); microRNA