Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages

Abstract

Background: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo.

Methods: To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6-7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count.

Results: The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM.

Conclusion: The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

Keywords: Cell persistence; Cell therapy; Induced pluripotent stem cell-derived cardiomyocytes.

Fig. 1

Decellularized ECM scaffold on a funnel dish. a Representative picture of a custom-made funnel dish, upper part left, and lower part right. b Schematic view of a funnel dish. c Decellularized ECM scaffold stained with Masson-Trichrome-Staining. Green: collagen, Red: keratin and muscle fibers, Pink: cytoplasm

Fig. 2

Adhesion of iPSC-CM to fibronectin-coated dishes. a Representative pictures of attached iPSC-CM 5 min (left panel) and 10 min (right panel) after plating on fibronectin-coated cell culture dishes. Scale bar 100 μm. b Number of attached iPSC-CM plated at different stages (14 days, 16 days, and 18 days after starting the differentiation process). 20,000 cells were plated in 100 μl drops per dish. Cells were photographed and counted 5 (square) or 10 (triangle) minutes after plating. All data are presented as mean + S.E.M. Statistical analysis was performed using one-way ANOVA with post-test, 5 min: p = 0.709; 10 min p = 0.326

Fig. 3

Adhesion of iPSC-CM to ventricular ECM scaffolds. a Representative picture of decellularized ventricular heart tissue (left) with attached eGFP-positive iPSC-CM (right). Scale bar 100 μm. b Absolute number of iPSC-CM detected by qPCR. iPSC-CM were added to ECM scaffolds at different time points after starting the differentiation process (14, 16, and 18 days). The number of attached cells was below the detection limit of the qPCR in 2/6 samples for iPSC-CM at day 14, 5/6 samples for iPSC-CM at day 16 and all 5 samples for iPSC-CM at day 18 (data points not included in the diagram)

Fig. 4

Cell persistence after transplantation of iPSC-CM in infarcted mouse hearts. a Representative image of eGFP-positive iPSC-CM within a ventricular slice of the recipient heart, 7 days after transplantation. Left scale bar 1 mm, right scale bar 100 μm. b Number of iPSC-CM assessed by qPCR 6–7 days after injecting 2 × 500,000 iPSC-CM in the infarct border zone. iPSC-CM were transplanted at 14 or 16 days after starting the differentiation process. Mean persistence was approximately 7-fold higher in cells transplanted at day 14 of differentiation compared to day 16 iPSC-CM. Statistical analysis was performed using the Mann–Whitney rank sum test, p < 0.001

Fig. 5

Functional integration of transplanted iPSC-CM into the myocardium. a Representative bright field image of a ventricular heart slice (left picture) with retained eGFP-positive iPSC-CM (right picture) 7 days after injection of 2 × 0.5 × 10⁶ day 16 iPSC-CM. Left scale bar 1 mm, right scale bar 100 μm. The positions of the stimulation electrode (SE) and the recordings in host tissue and iPSC-CM are marked. b The quality of electrical integration of day 14 iPSC-CM, day 16 iPSC-CM, and native host cardiomyocytes assessed by the maximal stimulation frequency without conduction block. c Representative action potentials of a host cardiomyocyte and transplanted iPSC-CM. d Action potential properties of transplanted day 14 iPSC-CM (n = 6), day 16 iPSC-CM (n = 8), and host cardiomyocytes (n = 8). MDP maximal diastolic potential, Vmax maximal upstroke velocity, APD50 action potential duration at 50% of repolarization, APD90 action potential duration at 90% of repolarization. e Statistical analyses of action potential properties. Statistical analysis were performed using one-way ANOVA with post-test for host vs. iPSC-CM and Student’s t test for day 14 vs. day 16 iPSC-CM