Demethylation of induced pluripotent stem cells from type 1 diabetic patients enhances differentiation into functional pancreatic β cells

Pharmacological preconditioning with volatile anesthetics (APC) protects the myocardium against ischemia/reperfusion injury via multiple pathways that include the cardiac sarcolemmal (sarc) KATP channel. However, APC is altered or absent in metabolic diseases such as diabetes. To investigate the underlying mechanisms we evaluated the expression and activity of cardiac sarcKATP channel in cardiomyocytes (CMs) differentiated from induced pluripotent stem cells of non-diabetic (N-iPSCs) and type II diabetic (DM-iPSCs) patients. Differentiation of human iPSCs into CMs was confirmed by positive immunostaining for cardiac-specific markers and expression of green fluorescent protein under transcriptional control of cardiac promoter myosin light chain-2v that was delivered by lentiviral vector. Immunocytochemical analysis of cardiac sarcKATP channel subunits was carried out with Anti-Kir6.2 and Anti-SUR2A antibodies. Images were captured using a laser-scanning confocal microscope. Single KATP channel activity was monitored from excised inside-out patches at membrane potentials +80 mV to -80 mV, in symmetrical 140 mM K+. The channel was closed by 1 µM glibenclamide and 2 mM ATP. Immunocytochemical analysis showed the presence and colocalization of Kir6.2 and SUR2A proteins in CMs derived from both N-iPSCs and DM-iPSCs. Interestingly, sarcolemmal expression of these subunits appeared lower in DM-iPSCs. Electrophysiological recordings demonstrated that at 5 µM ATP internal single sarcKATP channels were open more frequently in N-iPSCs (7 of 16 cultures) than in DM-iPSCs (3 of 16 cultures). When measured at +40 mV, the single channel current amplitude (2.2 pA) and conductance (55 pS) were typical for the cardiac sarcKATP channel in N-iPSCs. By contrast, the DM-iPSC channels flickered between the open and closed state, resulting in higher noise level and decreased current amplitude (1.6 pA). This is the first report of sarcKATP channel expression and activity in CMs differentiated from human N-iPSCs and DM-iPSCs. The study suggests that compromised APC in human diabetic heart may in part result from altered expression of sarcKATP channel subunits and altered channel kinetics.

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Study neurofibromatosis type 1 disease with patient-specific induced pluripotent stem cells

10.5353/th_b5760945 ◽

2016 ◽

Author(s):

Dajiang Qin

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Neurofibromatosis Type 1 ◽

Pluripotent Stem Cells ◽

Neurofibromatosis Type ◽

Patient Specific ◽

Induced Pluripotent

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The potential of human induced pluripotent stem cells for modelling diabetic wound healing in vitro

Clinical Science ◽

10.1042/cs20171483 ◽

2018 ◽

Vol 132 (15) ◽

pp. 1629-1643 ◽

Cited By ~ 4

Author(s):

Patricia E. Martin ◽

Erin M. O’Shaughnessy ◽

Catherine S. Wright ◽

Annette Graham

Keyword(s):

Stem Cells ◽

Wound Healing ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Chronic Wounds ◽

Healing Process ◽

Diabetic Patients ◽

Impaired Wound Healing ◽

Induced Pluripotent

Impaired wound healing and ulceration caused by diabetes mellitus, is a significant healthcare burden, markedly impairs quality of life for patients, and is the major cause of amputation worldwide. Current experimental approaches used to investigate the complex wound healing process often involve cultures of fibroblasts and/or keratinocytes in vitro, which can be limited in terms of complexity and capacity, or utilisation of rodent models in which the mechanisms of wound repair differ substantively from that in humans. However, advances in tissue engineering, and the discovery of strategies to reprogramme adult somatic cells to pluripotency, has led to the possibility of developing models of human skin on a large scale. Generation of induced pluripotent stem cells (iPSCs) from tissues donated by diabetic patients allows the (epi)genetic background of this disease to be studied, and the ability to differentiate iPSCs to multiple cell types found within skin may facilitate the development of more complex skin models; these advances offer key opportunities for improving modelling of wound healing in diabetes, and the development of effective therapeutics for treatment of chronic wounds.

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