Assessment of Induced Pluripotent Stem Cell-Derived Cardiomyocyte Contractility Using Micropost Arrays

Author(s):  
Marita L. Rodriguez ◽  
Charles E. Murry ◽  
Nathan J. Sniadecki

Cardiovascular stem cell therapies have shown increasing promise as a potential therapeutic means for reversing the effects of a myocardial infarction [1]. Out of the currently available sources of human stem cells, human induced pluripotent stem cells (hiPSCs) are very promising in that: the number of cell lines that can be induced to the pluripotent state is extremely vast, they serve as a potential source for patient-specific cardiomyocytes, and their use is non-controversial. However, before they can be used feasibly in a clinical setting, the functional engraftment of these cells into the host tissue must be improved [2]. It is hypothesized that the structural and functional maturity of the stem-cell derived cardiomyocytes prior to implantation, may significantly affect the ability of these cells to engraft with resident heart tissue [3]. One of the most important functional characteristics of a cardiomyocyte is its ability to produce contractile forces. However, assessing the contractile properties of single iPS-CMs is a difficult task. iPS-CMs generally have relatively unorganized cytoskeletons, with stress fibers in multiple directions. This trait renders one or two-point force assays ineffectual in determining total cell forces. Furthermore, iPS-CMs don’t spread well on tissue culture surfaces, which make two-dimensional force measurements almost impossible.

Circulation ◽  
2020 ◽  
Vol 142 (16) ◽  
pp. 1562-1578
Author(s):  
Alexandra Madsen ◽  
Grit Höppner ◽  
Julia Krause ◽  
Marc N. Hirt ◽  
Sandra D. Laufer ◽  
...  

Background: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell–derived cardiomyocytes. Methods: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell–derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. Results: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. Conclusion: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.


2019 ◽  
Vol 20 (8) ◽  
pp. 2026 ◽  
Author(s):  
Ramazzotti ◽  
Ratti ◽  
Fiume ◽  
Yung Follo ◽  
Billi ◽  
...  

Stem cells are undifferentiated cells that can give rise to several different cell types and can self-renew. Given their ability to differentiate into different lineages, stem cells retain huge therapeutic potential for regenerative medicine. Therefore, the understanding of the signaling pathways involved in stem cell pluripotency maintenance and differentiation has a paramount importance in order to understand these biological processes and to develop therapeutic strategies. In this review, we focus on phosphoinositide 3 kinase (PI3K) since its signaling pathway regulates many cellular processes, such as cell growth, proliferation, survival, and cellular transformation. Precisely, in human stem cells, the PI3K cascade is involved in different processes from pluripotency and induced pluripotent stem cell (iPSC) reprogramming to mesenchymal and oral mesenchymal differentiation, through different and interconnected mechanisms.


2017 ◽  
Vol 113 (5) ◽  
pp. 531-541 ◽  
Author(s):  
Marcella Rocchetti ◽  
Luca Sala ◽  
Lisa Dreizehnter ◽  
Lia Crotti ◽  
Daniel Sinnecker ◽  
...  

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