Cell persistence and electrical integration of transplanted fetal cardiomyocytes from different developmental stages

Cell replacement therapy is a promising approach to overcome thelimited regenerative potential of damaged myocardium. Persistenceand functional integration of transplanted cardiomyocytes are crucialfor a safe and efficient cell replacement therapy. The mechanismspromoting cell persistence and integration are poorly understood. Thedevelopmental stage of transplanted cells appears to be an importantfactor, since adult cardiomyocytes do not survive transplantation [1],while immature cardiomyocytes do survive and integrate functionally[1,2]. During fetal development, cardiomyocytes undergo substantialstructural and electrophysiological alterations [3–5], but it has notbeen studied yet, whether different stages of fetal developmentinfluence persistence and functional integration of transplantedcardiomyocytes, and if there is a distinct immature developmentalstagethatpromotesanoptimalpersistenceandfunctionoftransplantedcardiomyocytes.Therefore,weinvestigatedcellpersistenceandelectro-physiological integration of transplanted murine fetal cardiomyocytes(FCM) from different developmental stages.FetalventriclesfromtransgenicCrl:OF1miceexpressingeGFPundercontrol of the alpha-actin promoter were harvested at days 9.5(earlyFCM),14.5(interFCM)and18.5(lateFCM)p.c.Injectionsofdisso-ciated FCM were performed at two different sites of the left ventricularwall (2 × 450.000 cells) in adult Crl:OF1 mice. Six days after surgery,viable slices of recipient hearts (150 μm thickness) were producedwith a microtome (Leica, Germany). Intracellular action potentialswererecordedwithglassmicroelectrodes(WPI,USA).Sliceswerestim-ulated focally in host tissue. Cell preparation, surgery and microelec-trode recordings were performed as described before [2].Cellpersistence was quantified using a score ranging from “0” to “3”(0 = no graft cells visible; 1 = few and small clusters of transplantedcells (Fig. 1A + C); 2 = larger clusters of transplanted cells; 3 = largeareas of transplanted cells (Fig. 1B)). Quantitative analysis of cell persis-tence by quantitative TaqMan real-time PCR (qPCR) was performed sim-ilarlyasdescribedbefore [6,7].Sliceswerelysated,andgenomicDNAwasprepared, which was then used for qP CR with primers against the genesfor eGFP (grafted cells) and for beta-actin (total cell count). Data weretested for statistical significance by one-way ANOVA with post test or, ifnormality test failed, with Kruskal–Wallis test. Data are presentedas mean ± SEM. All experiments were approved by the local animalwelfare committee, and all animals received humane care.Persistence score was 2.91 ± 0.09 (n = 11) in interFCM, whichwas significantly higher compared to lateFCM (1.04 ± 0.12, n = 23,p b 0.001)and earlyFCM(0.40 ± 0.11,n = 20, p b 0.001vs.interFCM,p b 0.05 vs. lateFCM, Fig. 1D). The qPCR analysis confirmed thesefindings with 15,189 ± 4791 transgenic cells per mg detected inrecipient heart slices after injection of interFCM (n = 11), 3972 ±1730 cells/mg for lateFCM (n = 20, p b 0.05 vs. interFCM) and2290 ± 1851 cells/mg for earlyFCM (n = 17, p b 0.01 vs. interFCM,p = n.s. vs. lateFCM; Fig. 1E). Persistence of earlyFCM was insufficientfor electrophysiological analyses.The maximal stimulation frequency without conduction blocks,which was used to indicate the quality of integration, was8.62 ± 0.42 Hz in interFCM (n = 13) and 4.60 ± 0.67 Hz in lateFCM(n = 10, p b 0.001, Fig. 2A).Host cellsfollowed higherstimulation fre-quencies(11.00 ± 0.30 Hz;n = 21).Toquantifythevelocityofexcita-tion spread, we analyzed the delay between stimulus and actionpotential upstroke in host and graft cardiomyocytes. This delay waslower in interFCM (15.14 ± 1.03 ms, n = 18) than in lateFCM(28.49 ± 1.98 ms,n = 16,p b 0.001, Fig.2B + D),butwassignificant-ly higher in all transplanted FCM than in host cardiomyocytes(7.71 ± 0.21 ms, n = 28, pb 0.001 vs. interFCM and lateFCM). Thedistance between stimulation and recording electrode was equal inthese measurements (stimulation electrode to host cardiomyocytes1.51 ± 0.09 mm, to transplanted interFCM 1.55 ± 0.02 mm, totransplanted lateFCM 1.29 ± 0.16 mm; p = n.s.). Action potential