Human Mesenchymal Stem Cells as a Gene Delivery System to Create Cardiac Pacemakers

Abstract

Abstract-We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs ϩ -sensitive current (31.1Ϯ3.8 pA/pF at Ϫ150 mV) activating in the diastolic potential range with reversal potential of Ϫ37.5Ϯ1.0 mV, confirming the expressed current as I f -like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93Ϯ16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161Ϯ4 bpm when hMSCs were expressing both EGFPϩmHCN2 (PϽ0.05). We next injected 10 6 hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45Ϯ1 bpm, 2 of right-sided origin and 2 of left). In the EGFPϩmHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rateϭ61Ϯ5 bpm; PϽ0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.

Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes—A model

“…This result confirms the conjecture, based on results of experiments with tissues [7], [6], that a stem cell hMSC transfected with HCN2 gene can induce oscillations in a cardiac cell.…”

“…In experiments [6], stem cells added to cultured neonatal myocytes that are self-oscillatory. The stem cells decreased the oscillation period of these myocytes from T ∼ 660 to T ∼ 370ms.…”

“…In exploring such systems, we believe that an adequate combination of experiments with theoretical predictions can lead to a much faster results, by reaching a better quantitative understanding and in so doing, in reducing the number of experiments. To this end, one needs precise and adaptable mathematical models based on well-established concepts and results [6]- [23].…”

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

“…34 Recently, an electroporation-based technology called nucleofector, which elicits the destabilization of both cytoplasmic and nuclear membranes, has been proposed as an alternative tool for gene transfer. 47 Although MSC nucleofection resulted in high transfection efficiency rates (450%), [48][49][50] it unfortunately led to limited cell recovery, 51-53 reflecting a major drawback of most electroporation techniques employed to facilitate the transplantation of in vitro genetically modified cells.…”

Gene Therapy for Cardiac Arrhythmias

“…Mesenchymal stem cells (MSC) were also utilized in a similar strategy. 32,33 The injection of HCN2-overexpressed MSC into canine ventricles developed a spontaneous rhythm. In contrast to fibroblasts, MSC expresses gap junction channels, which enable a cell to cell coupling without polyethylene glycol to be obtained.…”