Decellularized Extracellular Matrix Powder Accelerates Metabolic Maturation at Early Stages of Cardiac Differentiation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

During fetal development, cardiomyocytes switch from glycolysis to oxidative metabolism to sustain the energy requirements of functional cells. State-of-the-art cardiac differentiation protocols yield phenotypically immature cardiomyocytes, and common methods to improve metabolic maturation require multistep protocols to induce maturation only after cardiac specification is completed. Here, we describe a maturation method using ventricle-derived decellularized extracellular matrix (dECM) that promoted early-stage metabolic maturation of cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). Chemically and architecturally preserved particles (45-500 µm) of pig ventricular dECM were added to hiPSCs at the start of differentiation. At the end of our maturation protocol (day 15 of cardiac differentiation), we observed an intimate interaction between cardiomyocytes and dECM particles without impairment of cardiac differentiation efficiency (approx. 70% of cTNT+). Compared with control cells (those cultured without pig dECM), 15-day-old dECM-treated cardiomyocytes demonstrated increased expression of markers related to cardiac metabolic maturation, MAPK1, FOXO1, and FOXO3, and a switch from ITGA6 (the immature integrin isoform) to ITGA3 and ITGA7 (those present in adult cardiomyocytes). Electrical parameters and responsiveness to dobutamine also improved in pig ventricular dECM-treated cells. Extending the culture time to 30 days, we observed a switch from glucose to fatty acid metabolism, indicated by decreased glucose uptake and increased fatty acid consumption in cells cultured with dECM. Together, these data suggest that dECM contains endogenous cues that enable metabolic maturation of hiPSC-CMs at early stages of cardiac differentiation.

Polymerized Laminin-521: A Feasible Substrate for Expanding Induced Pluripotent Stem Cells at a Low Protein Concentration.

Laminins (LNs) play a central role in the self-assembly and maintenance of basement membranes and are involved in critical interactions between cells and other extracellular matrix (ECM) proteins. Among the defined, xeno-free ECM culture matrices, LNs-namely LN521-have emerged as promising coating systems for the large-scale expansion of induced pluripotent stem cells (iPSCs). The biologic activity of LNs is enhanced by their acidification-induced self-polymerization into a cell-associated network called polylaminin (polyLN), which can recapitulate the native-like polymeric array in a cell-free system. Here, we show for the first time to our knowledge that polyLN521 displays a native-like hexagonal-like structure and that, at basal and low concentrations, it permits the large-scale expansion of human iPSCs. Human iPSCs expanded with polyLN521 maintained the pluripotent state and showed no impairment of karyotype stability or telomere length. These results suggest that low-concentration polyLN521 is a stable and cost-effective coating for large-scale iPSC expansion.

Mining the Mesenchymal Stromal Cell Secretome in Patients with Chronic Left Ventricular Dysfunction.

Close examination of the initial results of cardiovascular cell therapy clinical trials indicates the importance of patient-specific differences on outcomes and the need to optimize or customize cell therapies. The fields of regenerative medicine and cell therapy have transitioned from using heterogeneous bone marrow mononuclear cells (BMMNCs) to mesenchymal stromal cells (MSCs), which are believed to elicit benefits through paracrine activity. Here, we examined MSCs from the BMMNCs of heart failure patients enrolled in the FOCUS-CCTRN trial. We sought to identify differences in MSCs between patients who improved and those who declined in heart function, regardless of treatment received. Although we did not observe differences in the cell profile of MSCs between groups, we did find significant differences in the MSC secretome profile between patients who improved or declined. We conclude that "mining" the MSC secretome may provide clues to better understand the impact of patient characteristics on outcomes after cell therapy and this knowledge can inform future cell therapy trials.

Whole Heart Engineering: Advances and Challenges.

Bioengineering a solid organ for organ replacement is a growing endeavor in regenerative medicine. Our approach - recellularization of a decellularized cadaveric organ scaffold with human cells - is currently the most promising approach to building a complex solid vascularized organ to be utilized in vivo, which remains the major unmet need and a key challenge. The 2008 publication of perfusion-based decellularization and partial recellularization of a rat heart revolutionized the tissue engineering field by showing that it was feasible to rebuild an organ using a decellularized extracellular matrix scaffold. Toward the goal of clinical translation of bioengineered tissues and organs, there is increasing recognition of the underlying need to better integrate basic science domains and industry. From the perspective of a research group focusing on whole heart engineering, we discuss the current approaches and advances in whole organ engineering research as they relate to this multidisciplinary field's 3 major pillars: organ scaffolds, large numbers of cells, and biomimetic bioreactor systems. The success of whole organ engineering will require optimization of protocols to produce biologically-active scaffolds for multiple organ systems, and further technological innovation both to produce the massive quantities of high-quality cells needed for recellularization and to engineer a bioreactor with physiologic stimuli to recapitulate organ function. Also discussed are the challenges to building an implantable vascularized solid organ.

Restoring anatomical complexity of a left ventricle wall as a step toward bioengineering a human heart with human induced pluripotent stem cell-derived cardiac cells.

The heart is a highly complex, multicellular solid organ with energy-demanding processes that require a dense vascular network, extensive cell-cell interactions, and extracellular matrix (ECM)-mediated crosstalk among heterogeneous cell populations. Here, we describe the regeneration of left ventricular (LV) wall using decellularized whole rabbit heart scaffolds recellularized exclusively with human induced pluripotent stem cell-derived endothelial cells, cardiomyocytes, and other cardiac cell types. Cells were sequentially delivered to the scaffold using an optimized endothelial cell:cardiomyocyte media. Macroscopic assessment after 60 days showed that the LV wall of recellularized hearts was anatomically restored to full thickness from base to apex and endocardium to epicardium. Histologic analysis of the recellularized LV wall revealed a heterogeneous pool of cardiac cells containing aligned cardiac troponin T-positive cells in close contact with ECM; vessels varied from large artery-like, surrounded by smooth muscle actin+ cells, to capillary-like. Vessel patency was demonstrated after perfusion of recellularized hearts transplanted into the femoral artery bed of a pig. The construct exhibited visible beating and responded to chronotropic drug administration. These results demonstrate the ability to tissue engineer a vascularized, full-thickness LV wall with an unparalleled level of microanatomical organization and multicellular composition, using decellularized ECM and human cardiomyocytes, endothelial cells, and other cardiac cell types. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (ECM) is a bioactive template for tissue engineering, but recellularizing acellular whole heart scaffolds is challenging. Here, we successfully revascularized and repopulated a large, full-thickness portion of a ventricle using human induced pluripotent stem cell-derived endothelial and cardiac cells. At 60 days, histologic studies showed that the microanatomical organization and cellular composition of this region was similar to that of the native heart. The recellularized heart showed visible beating and responded appropriately to heartbeat-altering drugs. Vessels surrounded by smooth muscle cells and endothelial cells supported blood flow through the vessels of a recellularized heart that was surgically connected to a pig femoral artery. These findings move this approach closer to the possibility of clinical translation.

Characterization of perfusion decellularized whole animal body, isolated organs, and multi-organ systems for tissue engineering applications.

To expand the application of perfusion decellularization beyond isolated single organs, we used the native vasculature of adult and neonatal rats to systemically decellularize the organs of a whole animal in situ. Acellular scaffolds were generated from kidney, liver, lower limb, heart-lung system, and a whole animal body, demonstrating that perfusion decellularization technology is applicable to any perfusable tissue, independent of age. Biochemical and histological analyses demonstrated that organs and organ systems (heart-lung pair and lower limb) were successfully decellularized, retaining their extracellular matrix (ECM) structure and organ-specific composition, as evidenced by differences in organ-specific scaffold stiffness. Altogether, we demonstrated that organs, organ systems and whole animal bodies can be perfusion decellularized while retaining ECM components and biomechanics.

Cues from human atrial extracellular matrix enrich the atrial differentiation of human induced pluripotent stem cell-derived cardiomyocytes.

New robust and reproducible differentiation approaches are needed to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes of specific subtypes in predictable quantities for tissue-specific disease modeling, tissue engineering, and eventual clinical translation. Here, we assessed whether powdered decellularized extracellular matrix (dECM) particles contained chamber-specific cues that could direct the cardiac differentiation of human iPSCs toward an atrial phenotype. Human hearts were dissected and the left ventricle (LV) and left atria (LA) were isolated, minced, and decellularized using an adapted submersion decellularization technique to generate chamber-specific powdered dECM. Comparative proteomic analyses showed chamber-specific dECM segregation, with atrial- and ventricle-specific proteins uniquely present in powdered dECM-hA and dECM-hV, respectively. Cell populations differentiated in the presence of dECM-hA showed upregulated atrial molecular markers and a two-fold increase in the number of atrial-like cells as compared with cells differentiated with dECM-hV or no dECM (control). Finally, electrophysiological data showed an increase in action potentials characteristic of atrial-like cells in the dECM-hA group. These findings support the hypothesis that dECM powder derived from human atria retained endogenous cues to drive cardiac differentiation toward an atrial fate.

Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System.

The number of high-quality cells required for engineering an adult human-sized bioartificial organ is greater than one billion. Until the emergence of induced pluripotent stem cells (iPSCs), autologous cell sources of this magnitude and with the required complexity were not available. Growing this number of cells in a traditional 2D cell culture system requires extensive time, resources, and effort and does not always meet clinical requirements. The use of a closed cell culture system is an efficient and clinically applicable method that can be used to expand cells under controlled conditions. We aimed to use the Quantum Cell Expansion System (QES) as an iPSC monolayer-based expansion system. Human iPSCs were expanded (up to 14-fold) using the QES on two different coatings (laminin 521 (LN521) and vitronectin (VN)), and a karyotype analysis was performed. The cells were characterized for spontaneous differentiation and pluripotency by RT-PCR and flow cytometry. Our results demonstrated that the QES provides the necessary environment for exponential iPSC growth, reaching 689.75 × 106 ± 86.88 × 106 in less than 7 days using the LN521 coating with a population doubling level of 3.80 ± 0.19. The same result was not observed when VN was used as a coating. The cells maintained normal karyotype (46-XX), expressed pluripotency markers (OCT4, NANOG, LIN28, SOX2, REX1, DPPA4, NODAL, TDGFb, TERT3, and GDF), and expressed high levels of OCT4, SOX2, NANOG, SSEA4, TRA1-60, and TRA1-81. Spontaneous differentiation into ectoderm (NESTIN, TUBB3, and NEFH), mesoderm (MSX1, BMP4, and T), and endoderm (GATA6, AFP, and SOX17) lineages was detected by RT-PCR with both coating systems. We conclude that the QES maintains the stemness of iPSCs and is a promising platform to provide the number of cells necessary to recellularize small human-sized organ scaffolds for clinical purposes.