Abstract 247: Expression and Activity of K ATP Channel in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells of Nondiabetic and Type II Diabetic Patients

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Scott Canfield ◽  
Zeljko J Bosnjak ◽  
Anna Stadnicka
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Katp Channel ◽  
Current Amplitude ◽  
Diabetic Patients ◽  
Type Ii ◽  
Immunocytochemical Analysis ◽  
Induced Pluripotent ◽  
Expression And Activity

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.

scholarly journals MicroRNA-22 enhances the differentiation of mouse induced pluripotent stem cells into alveolar epithelial type II cells

2020 ◽  
Vol 64 (s2) ◽  
Author(s):  
Fan Tao ◽  
Feng Wang ◽  
Weichen Zhang ◽  
Yaming Hao
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Lung Surfactant ◽  
Lung Epithelial Cells ◽  
Type Ii ◽  
Alveolar Epithelial ◽  
Cellular Processes ◽  
Cell Based Therapy ◽  
Induced Pluripotent

Considerable evidence has verified that microRNAs (miRNAs) play important roles in various cellular processes including differentiation. However, the regulatory roles of miRNAs involved in the differentiation of induced pluripotent stem cells (iPSC) into lung epithelial cells are still unknown. In this study, we first evaluated the current protocols to differentiate iPSC into alveolar epithelial type II (AEC II) cells, but the efficiency is low. We next identified that miR-22 can efficiently enhance the differentiation of iPSC into AEC II cells under the stimulation of proper growth factors and growing on appropriate matrix. Moreover, the AEC II cells generated from iPSC with miR-22 overexpression can proliferate and secrete lung surfactant. Here, we discovered a previously unknown interaction between miR-22 and iPSC differentiation but also provide a potential target for the effective derivation of AEC II from iPSCs for cell-based therapy.

scholarly journals Efficient Differentiation of Mouse Induced Pluripotent Stem Cells into Alveolar Epithelium Type II with a BRD4 Inhibitor

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Toru Momozane ◽  
Eriko Fukui ◽  
Soichiro Funaki ◽  
Makoto Fujii ◽  
Yuhei Kinehara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Protein C ◽  
Three Dimensional ◽  
Alveolar Epithelium ◽  
Type Ii ◽  
Differentiation Induction ◽  
Induced Pluripotent ◽  
Differentiation Efficiency

Regenerative medicine has continued to progress for lung biology and lung diseases. Efforts have focused on a variety of different applications for pluripotent stem cells. Several groups have reported successful methods for inducing differentiation of induced pluripotent stem cells (iPSCs) into the airway epithelium such as alveolar epithelium type II (ATII). However, differentiation efficiency varies among reports and improvements are needed. In the present paper, we propose a novel method for elimination of residual undifferentiated murine iPSCs using JQ1, a potent inhibitor of bromodomain (BRD) and extraterminal domain (BET) family proteins, for efficient differentiation into ATII. First, the murine iPSC line 20D-17 was induced to differentiate into ATII over a period of 26 days (days 0-26) using previously reported embryoid body seeding and stepwise differentiation methods. mRNA expressions of differentiation markers including surfactant protein C (Sftpc) were confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) results, and 17% of the cells were shown positive for prosurfactant protein C (proSPC) in flow cytometry analysis. Next, those cells were cultured three-dimensionally in Matrigel for an additional 14 days (days 26-40), during which JQ1 was added for 4 days (days 28-32) to remove residual undifferentiated iPSCs. As a result, on day 40, the mRNA expression level of Sftpc in the three-dimensional culture was maintained at the same level as on day 26 and shown to be further increased by the addition of JQ1, with 39% of the cells found to express proSPC, showing that differentiation efficiency could be further increased. Three-dimensional culture with BRD4 inhibition by JQ1 improved the differentiation induction efficiency to ATII by removing residual undifferentiated murine iPSCs during the differentiation induction process.

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

2018 ◽  
Vol 132 (15) ◽  
pp. 1629-1643 ◽  
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.

Differentiation of Urine-Derived Human Induced Pluripotent Stem Cells to Alveolar Type II Epithelial Cells

2016 ◽  
Vol 18 (1) ◽  
pp. 30-36 ◽  
Author(s):  
Chen Wang ◽  
Feilong Hei ◽  
Zhihai Ju ◽  
Jie Yu ◽  
Shengnan Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Epithelial Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Alveolar Type ◽  
Type Ii ◽  
Induced Pluripotent

scholarly journals RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome

2019 ◽  
Vol 40 (37) ◽  
pp. 3081-3094 ◽  
Author(s):  
Nadjet Belbachir ◽  
Vincent Portero ◽  
Zeina R Al Sayed ◽  
Jean-Baptiste Gourraud ◽  
Florian Dilasser ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Brugada Syndrome ◽  
Susceptibility Gene ◽  
Focal Adhesions ◽  
Current Amplitude ◽  
Structural Investigations ◽  
Human Cardiomyocytes ◽  
Induced Pluripotent

Abstract Aims The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. Methods and results Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. Conclusion This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

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