Bioengineering of A Humanized Heart by Seeding of hiPSC-Derived Cardiovascular Progenitor Cells into Growth Factor-tethered Rat Heart Matrix

Background: Millions of people worldwide suffer from cardiovasculardiseases. Although current interventional and pharmacologicalapproaches provide efficient therapies, curativetreatment of end-stage heart failure is limited to heart transplantation.Bioengineering of whole hearts using human embryonicstem cells (hESCs)-derived cardiovascular progenitorcells (CPCs) and natural matrices is a promising approach toovercome organ donor shortage threatening millions of patientswaiting for heart transplantation.Materials and Methods: Here, we developed a novel strategyfor generation of heart constructs by repopulating engineereddecellularized rat hearts using hESCs-derived CPCs. we modifieddecellularization protocol to improve efficacy which wasconfirmed by multiple tests including DNA content analysisas well as biochemical studies. The decellularized hearts wererecellularized by hESC-derived CPCs, which were generatedin a scalable suspension bioreactor system. To improve CPCsproliferation and differentiation, we immobilized bFGF ontoheart ECM prior to cell perfusion. Further optimization of seedingdensity and loading intervals allowed uniform recellularizationof the heart scaffold. At day ۱۲ post seeding, functionalstudies were performed on recellularized hearts. The beatingrhythm was evaluated using a multielectrode array system.Contraction motions were recorded using video microscopyand analyzed using a custom-made mathlab macro. qRTPCRand immunostaining was performed for cardiac specific markers.In-depth examination of the ultrastructure of seeded CPCsand CPC-derived cells were investigated by transmission electronmicroscopy (TEM).Results: We demonstrated that perfusion-decellularization ofwhole heart allows the generation of a heart ECM scaffold witha perfusable vascular tree and intact ۳D architecture, which actsas an efficient template to generate synchronously beating hearttissue. Comprehensive characterization of the decellularizedheart matrix demonstrated preservation of complex ECM proteins,۳D spatial orientation and the micro-structure of nativeheart. Careful expansion of CPCs in a scalable stirred-suspensionbioreactor combined with step-wise seeding (۶۰ millioncells in ۳ steps of ۲۰ million per ۱.۵ hour) onto decellularizedhearts containing immobilized bFGF resulted in improved retentionof CPCs and differentiation to cardiomyocytes, smoothmuscle cells and endothelial cells as evaluated by immunohistochemistryand qRT-PCR. We observed spontaneous andsynchronous contractions of humanized hearts after ۱۲ days ofperfusion as well as advanced alignment of myofilamentsConclusion: While clinical implementation of engineered hearttissues is recently examining in clinical trials, the whole heartbioengineering science is evolving quickly in order to circumventthe heart transplantation obstacles in patients with endstage heart failure. Nevertheless, heart organogenesis via decellularization/recellularization is still facing multiple technologicalchallenges before commercialization. Selection and largescale production of clinical grade starting cell ingredients, supplyingheart natural scaffold, and more importantly improvingcell repopulation procedure efficacy as well as the functionalityof lab grown hearts are the main challenges which need to beaddressed. Our study provides a robust platform for generationof artificial human hearts and resolves major bottlenecks hinderingfurther development of this technology. Bioengineeredhearts might soon find their way toward clinical application.

کلیدواژه ها:

Bioengineering ، bFGF ، Heart