Inducible Differentiation of iPS-Derived Cardiomyocyte Precursor Cells into Cardiomyocytes Using Biomimetic shRNA Technology

Objectives: Somatic cell therapy for myocardial regeneration has largely failed, and it has become clear that cardiomyocyte-like cells need to be used in order to achieve a sustained improvement of contractile function. However, mature cardiomyocytes (CM) do not proliferate, are susceptible to ischemia and poorly retained in recipient myocardium. Transplantation of robust, proliferative but committed precursor cells (CPC) may solve these problems. We sought to create CPCs for implantation in the failing myocardium that, once they have settled, consolidated, and proliferated in situ, can be selectively induced to mature.

Methods and Results: We designed an inducible customized biomimetic “small hairpin” shRNAs targeting the Wnt agonist β-catenin, integrated in a vector that also encodes for a fluorescent reporter (mCherry) and under the control of a doxycycline-inducible promoter. Wnt promotes self-renewal of cardiac progenitors and inhibits maturation towards CM, and Wnt modulation is widely used to differentiate hiPSC into cardiomyocytes. After establishing the system in HeLa cells, we transferred it to neonatal Sca-1+ hCPC, to directly reprogrammed iCMPs, and to the Opti-OX cell reprogramming platform, a commercial hiPS cell line designed to receive any transgene in the safe harbor locus AAVS1, which can be subsequently activated in trans upon doxycycline administration. Transfection using a wide range of “lipofection agents” proved unsatisfactory. The lentivirus-based “all-in-one transgene” displayed tight control of shRNA expression, negligible cell toxicity, robust shRNA/reporter gene expression (75–85% mCherry+ cells), and high Wnt-knockdown efficiencies (up to 70%) in HeLa cells after a 3-day doxycycline treatment. In primary human cells as well as in the OPTi-OX platform, we achieved a significant reduction of β-catenin correlating with reporter expression, and no cell toxicity upon activation. So far, the overall induced maturation efficiency in vitro ranked OPTi-OX < Sca-1+ CPC < iCMP.

Conclusion: An inducible system for targeted β-catenin downregulation by shRNA allows for significant and sustained Wnt signaling modulation. It can be transferred to human cardiomyocyte precursor cells so as to control cardiomyocyte differentiation via doxycycline delivery. This technology may be implemented to mature CPC/iCMP/iPSC in situ, after transplantation in the failing myocardium, opening new prospects for future cardiac cell therapy.

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