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. 2017 Oct;12(10):2097-2109.
doi: 10.1038/nprot.2017.089. Epub 2017 Sep 7.

Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells

Gun-Sik Cho  1   2 , Emmanouil Tampakakis  1 , Peter Andersen  1 , Chulan Kwon  1
Affiliations

Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells

Gun-Sik Cho et al. Nat Protoc. 2017 Oct.

Abstract

Pluripotent stem cells (PSCs), including induced PSCs, hold great potential for personalized disease modeling, drug testing and cell-based therapeutics. However, cells differentiated from PSCs remain immature in a dish, and thus there are serious caveats to their use in modeling adult-onset diseases such as cardiomyopathies and Alzheimer's disease. By taking advantage of knowledge gained about mammalian development and from bioinformatics analyses, we recently developed a neonatal rat system that enables maturation of PSC-derived cardiomyocytes into cardiomyocytes analogous to those seen in adult animals. Here we describe a detailed protocol that describes how to initiate the in vitro differentiation of mouse and human PSCs into cardiac progenitor cells, followed by intramyocardial delivery of the progenitor cells into neonatal rat hearts, in vivo incubation and analysis. The entire process takes ∼6 weeks, and the resulting cardiomyocytes can be analyzed for morphology, function and gene expression. The neonatal system provides a valuable tool for understanding the maturation and pathogenesis of adult human heart muscle cells, and this concept may be expanded to maturing other PSC-derived cell types, including those containing mutations that lead to the development of diseases in the adult.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

Authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Experimental protocol for in vivo and in vitro cardiomyocyte maturation. Left: Schematic illustration showing in vitro differentiation of mouse Isl1+ CPCs in red and human Isl1+ CPCs in green. Right: in vitro vs. in vivo maturation of cardiomyocytes derived from mouse and human-PSCs.
Figure 2
Figure 2
Isl1 positive cardiac progenitor cells. A. Isl1-Cre driven expression of Red Fluorescent Protein (RFP) in a mouse embryoid body. B. RFP+ CPCs are purified by FACS. (62,000 live cells were sorted 14,410 of those were Isl1-positive (23.3%)). C. Immunostaining for Isl1 and flow cytometry analysis of fixed human CPCs. Left plot: negative control with only secondary antibody. (2384 cells were analyzed and of those 31 were only Isl1-positive (1.3%)). Right plot: immunostaining with Isl1 primary antibody. (2750 cells were analyzed and of those 1940 were Isl1-positive (70.3%)). Scale bar 50 μm.
Figure 3
Figure 3
Surgical transplantation of cardiac progenitor cells for in vivo maturation. A. Neonatal rats 1–7 days old are placed on ice to induce anesthesia. B. Use the fine scissors to make a ~5 mm incision in the skin between the third and fourth ribs and gently separate the skin from the underlying muscles using fine forceps. Dissect the muscles of the intercostal space to create an opening of ~4 mm using the forceps C. Inject CPCs to the left ventricular apex using a microneedle connected to the Eppendorf FemtoJet Microinjector. D. Pinch the edges of the incision with the forceps and no more than 5 μL of the tissue adhesive glue to seal it the incision.
Figure 4
Figure 4
Isolation of mESC-CMs from injected rat hearts. A. Isolated rat hearts with injected RFP+ mouse CMs (arrow). Scale bars 500mm. B. Isolated adult-like mESC-CMs after processing of injected rat heart. The injected mESC-CMs are marked with GFP. Scale bars 100 μm.
Figure 5
Figure 5
In vivo matured mouse and human PSC-cardiomyocytes. Injected mouse and human cardiomyocytes were identified by GFP expression. In vivo matured CMs had sarcomere structure, size and binucleation very similar to adult CMs. cTnT: cardiac Troponin T. For all animal experiments appropriate institutional regulatory board permission was obtained. Scale bars 25 μm.
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