Sven Baumgartner

Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells

AIMS Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as promising cell type for cardiac cell replacement therapy. We investigated long-term electrophysiological integration and maturation of transplanted iPSCM, which are essential for therapeutic benefit. METHODS AND RESULTS Murine iPSCM expressing enhanced green fluorescent protein and a puromycin resistance under control of the α-myosin heavy chain promoter were purified by antibiotic selection and injected into adult mouse hearts. After 6-12 days, 3-6 weeks, or 6-8 months, viable slices of recipient hearts were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue, and intracellular action potentials (APs) were recorded with glass microelectrodes in transplanted cells and neighbouring host tissue within the slices. Persistence and electrical integration of transplanted iPSCM into recipient hearts could be demonstrated at all time points. Quality of coupling improved, as indicated by a maximal stimulation frequency without conduction blocks of 5.77 ± 0.54 Hz at 6-12 days, 8.98 ± 0.38 Hz at 3-6 weeks and 10.82 ± 1.07 Hz at 6-8 months after transplantation. AP properties of iPSCM became more mature from 6-12 days to 6-8 months after transplantation, but still differed significantly from those of host APs. CONCLUSION Transplanted iPSCM can persist in the long term and integrate electrically into host tissue, supporting their potential for cell replacement therapy. Quality of electrical integration improves between 6-12 days and 6-8 months after transplantation, and there are signs of an electrophysiological maturation. However, even after 6-8 months, AP properties of transplanted iPSCM differ from those of recipient cardiomyocytes.

Electrophysiological and morphological maturation of murine fetal cardiomyocytes during electrical stimulation in vitro.

The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of α-Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD50 and APD90) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD50 observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation.

Direct contact of umbilical cord blood endothelial progenitors with living cardiac tissue is a requirement for vascular tube-like structures formation.

The umbilical cord blood derived endothelial progenitor cells (EPCs) contribute to vascular regeneration in experimental models of ischaemia. However, their ability to participate in cardiovascular tissue restoration has not been elucidated yet. We employed a novel coculture system to investigate whether human EPCs have the capacity to integrate into living and ischaemic cardiac tissue, and participate to neovascularization. EPCs were cocultured with either living or ischaemic murine embryonic ventricular slices, in the presence or absence of a pro‐angiogenic growth factor cocktail consisting of VEGF, IGF‐1, EGF and bFGF. Tracking of EPCs within the cocultures was performed by cell transfection with green fluorescent protein or by immunostaining performed with anti‐human vWF, CD31, nuclei and mitochondria antibodies. EPCs generated vascular tube‐like structures in direct contact with the living ventricular slices. Furthermore, the pro‐angiogenic growth factor cocktail reduced significantly tubes formation. Coculture of EPCs with the living ventricular slices in a transwell system did not lead to vascular tube‐like structures formation, demonstrating that the direct contact is necessary and that the soluble factors secreted by the living slices were not sufficient for their induction. No vascular tubes were formed when EPCs were cocultured with ischaemic ventricular slices, even in the presence of the pro‐angiogenic cocktail. In conclusion, EPCs form vascular tube‐like structures in contact with living cardiac tissue and the direct cell‐to‐cell interaction is a prerequisite for their induction. Understanding the cardiac niche and micro‐environmental interactions that regulate EPCs integration and neovascularization are essential for applying these cells to cardiovascular regeneration.

Time-course of the electrophysiological maturation and integration of transplanted cardiomyocytes

Electrophysiological maturation and integration of transplanted cardiomyocytes are essential to enhance safety and efficiency of cell replacement therapy. Yet, little is known about these important processes. The aim of our study was to perform a detailed analysis of electrophysiological maturation and integration of transplanted cardiomyocytes. Fetal cardiomyocytes expressing enhanced green fluorescent protein were transplanted into cryoinjured mouse hearts. At 6, 9 and 12 days after transplantation, viable slices of recipient hearts were prepared and action potentials of transplanted and host cardiomyocytes within the slices were recorded by microelectrodes. In transplanted cells embedded in healthy host myocardium, action potential duration at 50% repolarization (APD50) decreased from 32.2 ± 3.3 ms at day 6 to 27.9 ± 2.6 ms at day 9 and 19.6 ± 1.6 ms at day 12. The latter value matched the APD50 of host cells (20.5 ± 3.2 ms, P=0.78). Integration improved in the course of time: 26% of cells at day 6 and 53% at day 12 revealed no conduction blocks up to a stimulation frequency of 10 Hz. APD50 was inversely correlated to the quality of electrical integration. In transplanted cells embedded into the cryoinjury, which showed no electrical integration, APD50 was 49.2 ± 4.3 ms at day 12. Fetal cardiomyocytes transplanted into healthy myocardium integrate electrically and mature after transplantation, their action potential properties after 12 days are comparable to those of host cardiomyocytes. Quality of electrical integration improves over time, but conduction blocks still occur at day 12 after transplantation. The pace of maturation correlates with the quality of electrical integration. Transplanted cells embedded in cryoinjured tissue still possess immature electrophysiological properties after 12 days.

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