Tissue engineered heart repair is developing rapidly, but needs refinement before clinical translation. We tested the hypothesis that force generating human engineered heart muscle (EHM) can be enhanced by integration of insulin-like growth factor-1 (IGF-1) secreting fibroblasts.
Methods: TetOn lentiviral particles encoding for IGF-1 and the Tet-transactivator (tTA) were cloned and used to stably transduce human foreskin fibroblasts (HFF). Baseline and doxycycline induced IGF-1 release from HFFTetOn+IGF1 was quantified by ELISA. HFFwt and HFFTetOn+IGF1 conditioned medium was layered over human embryonic stem cell (HES2) derived cardiomyocytes followed by an analysis of AKT-phosphorylation. EHMs were assembled from HES2-derived cardiomyocytes and HFF (HFFwt or HFFTetOn+IGF1) at 70:30 ratio. Transgene activation was induced by addition of doxycycline (10 ng/ml) for 7 days. Twitch forces and response to pharmacological stimuli were measured to assess the functional consequences of IGF-1 release. EHMs were subsequently subjected to morphological analysis or dissociated into single cells to assess cellular composition of EHMs.
Results: HFFTetOn+IGF1 released IGF-1 upon doxycycline stimulation (3.3x10-6 vs 8.3x10-8 [HFFwt] ng/ml/cell/day. Secreted IGF-1 from HFFTetOn+IGF1 induced Akt phosphorylation in HES2-derived cardiomyocytes (2.4±0.6 fold increase of [HFFwt]; n=3). EHMs with HFFTetOn+IGF1 developed significantly higher twitch forces than EHMs with HFFwt (0.24±0.03 vs 0.15±0.02 mN; n=10) under baseline conditions (IGF-1 leak). Doxycycline induced IGF-1 release further enhanced (P<0.05) EHM twitch force (0.26±0.03 mN; n=10). Single cell analysis from EHMs demonstrated cardiomyocyte hypertrophy in response to paracrine IGF-1 release (154±11% of [HFFwt] control; n=4). Histological analyses demonstrated that HFFTetOn+IGF1 supplemented EHMs contained thicker muscle bundles and enlarged cardiomyocytes.
Conclusion: EHM can be functionally enhanced by integration of drug-controllable IGF-1 release. Drug controllable, cell based paracrine release of protective factors may not only be exploited to enhance tissue engineered myocardium in vitro but also to achieve better survival and integration of EHM grafts in vivo.