| Autor: |
Lewis-Israeli YR; Institute for Quantitative Health Science and Engineering, Division of Developmental and Stem Cell Biology, Michigan State University; Department of Biomedical Engineering, College of Engineering, Michigan State University., Volmert BD; Institute for Quantitative Health Science and Engineering, Division of Developmental and Stem Cell Biology, Michigan State University; Department of Biomedical Engineering, College of Engineering, Michigan State University., Gabalski MA; Institute for Quantitative Health Science and Engineering, Division of Developmental and Stem Cell Biology, Michigan State University; Department of Biomedical Engineering, College of Engineering, Michigan State University., Huang AR; Institute for Quantitative Health Science and Engineering, Division of Developmental and Stem Cell Biology, Michigan State University; Department of Biomedical Engineering, College of Engineering, Michigan State University., Aguirre A; Institute for Quantitative Health Science and Engineering, Division of Developmental and Stem Cell Biology, Michigan State University; Department of Biomedical Engineering, College of Engineering, Michigan State University; aaguirre@msu.edu. |
| Abstrakt: |
The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs. The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues. |
