Roberto Gaetani

Cardiac tissue engineering using tissue printing technology and human cardiac progenitor cells.

Tissue engineering is emerging as a potential therapeutic approach to overcome limitations of cell therapy, like cell retention and survival, as well as to mechanically support the ventricular wall and thereby prevent dilation. Tissue printing technology (TP) offers the possibility to deliver, in a defined and organized manner, scaffolding materials and living cells. The aim of our study was to evaluate the combination of TP, human cardiac-derived cardiomyocyte progenitor cells (hCMPCs) and biomaterials to obtain a construct with cardiogenic potential for in vitro use or in vivo application. With this approach, we were able to generate an in vitro tissue with homogenous distribution of cells in the scaffold. Cell viability was determined after printing and showed that 92% and 89% of cells were viable at 1 and 7 days of culturing, respectively. Moreover, we demonstrated that printed hCMPCs retained their commitment for the cardiac lineage. In particular, we showed that 3D culture enhanced gene expression of the early cardiac transcription factors Nkx2.5, Gata-4 and Mef-2c as well as the sarcomeric protein TroponinT. Printed cells were also able to migrate from the alginate matrix and colonize a matrigel layer, thereby forming tubular-like structures. This indicated that printing can be used for defined cell delivery, while retaining functional properties.

Cardiac stem cells: isolation, expansion and experimental use for myocardial regeneration

Cellular cardiomyoplasty (myogenic cell grafting) is actively being explored as a novel method to regenerate damaged myocardium. The adult human heart contains small populations of indigenous committed cardiac stem cells or multipotent cardiac progenitor cells, identified by their cell-surface expression of c-kit (the receptor for stem cell factor), P-glycoprotein (a member of the multidrug resistance protein family), and Sca-1 (stem cell antigen 1, a mouse hematopoietic stem cell marker) or a Sca-1-like protein. Cardiac stem cells represent a logical source to exploit in cardiac regeneration therapy because, unlike other adult stem cells, they are likely to be intrinsically programmed to generate cardiac tissue in vitro and to increase cardiac tissue viability in vitro. Cardiac stem cell therapy could, therefore, change the fundamental approach to the treatment of heart disease.

Differentiation of human adult cardiac stem cells exposed to extremely low-frequency electromagnetic fields

AIMS Modulation of cardiac stem cell (CSC) differentiation with minimal manipulation is one of the main goals of clinical applicability of cell therapy for heart failure. CSCs, obtained from human myocardial bioptic specimens and grown as cardiospheres (CSps) and cardiosphere-derived cells (CDCs), can engraft and partially regenerate the infarcted myocardium, as previously described. In this paper we assessed the hypothesis that exposure of CSps and CDCs to extremely low-frequency electromagnetic fields (ELF-EMFs), tuned at Ca2+ ion cyclotron energy resonance (Ca2+-ICR), may drive their differentiation towards a cardiac-specific phenotype. METHODS AND RESULTS A significant increase in the expression of cardiac markers was observed after 5 days of exposure to Ca2+-ICR in both human CSps and CDCs, as evidenced at transcriptional, translational, and phenotypical levels. Ca2+ mobilization among intracellular storages was observed and confirmed by compartmentalized analysis of Ca2+ fluorescent probes. CONCLUSIONS These results suggest that ELF-EMFs tuned at Ca2+-ICR could be used to drive cardiac-specific differentiation in adult cardiac progenitor cells without any pharmacological or genetic manipulation of the cells that will be used for therapeutic purposes.

Concise Review: Heart Regeneration and the Role of Cardiac Stem Cells

Acute myocardial infarction leads to irreversible loss of cardiac myocytes, thereby diminishing the pump function of the heart. As a result, the strenuous workload imposed on the remaining cardiac myocytes often gives rise to subsequent cell loss until the vicious circle ends in chronic heart failure (CHF). Thus, we are in need of a therapy that could ameliorate or even reverse the disease progression of CHF. Endogenous regeneration of the mammalian heart has been shown in the neonatal heart, and the discovery that it may still persist in adulthood sparked hope for novel cardioregenerative therapies. As the basis for cardiomyocyte renewal, multipotent cardiac stem/progenitor cells (CSCs) that reside in the heart have been shown to differentiate into cardiac myocytes, smooth muscle cells, and vascular endothelial cells. These CSCs do have the potential to actively regenerate the heart but clearly fail to do so after abundant and segmental loss of cells, such as what occurs with myocardial infarction. Therefore, it is vital to continue research for the most optimal therapy based on the use or in situ stimulation of these CSCs. In this review, we discuss the current status of the cardioregenerative field. In particular, we summarize the current knowledge of CSCs as the regenerative substrate in the adult heart and their use in preclinical and clinical studies to repair the injured myocardium.

Human cardiosphere-seeded gelatin and collagen scaffolds as cardiogenic engineered bioconstructs

Cardiac tissue engineering (CTE) aims at regenerating damaged myocardium by combining cells to a biocompatible and/or bioactive matrix. Collagen and gelatin are among the most suitable materials used today for CTE approaches. In this study we compared the structural and biological features of collagen (C-RGD) or gelatin (G-FOAM)-based bioconstructs, seeded with human adult cardiac progenitor cells in the form of cardiospheres (CSps). The different morphology between C-RGD (fibrous ball-of-thread-like) and G-FOAM (trabecular sponge-like) was evidenced by SEM analysis and X-ray micro-tomography, and was reflected by their different mechanical characteristics. Seeded cells were viable and proliferating after 1 week in culture, and a reduced expression of cell-stress markers versus standard CSp culture was detected by realtime PCR. Cell engraftment inside the scaffolds was assessed by SEM microscopy and histology, evidencing more relevant cell migration and production of extracellular matrix in C-RGD versus G-FOAM. Immunofluorescence and realtime PCR analysis showed down-regulation of vascular and stemness markers, while early-to-late cardiac markers were consistently and significantly upregulated in G-FOAM and C-RGD compared to standard CSps culture, suggesting selective commitment towards cardiomyocytes. Overall our results suggest that CSp-bioconstructs have suitable mechanical properties and improved survival and cardiogenic properties, representing promising tools for CTE.

Evidence for Mechanisms Underlying the Functional Benefits of a Myocardial Matrix Hydrogel for Post-MI Treatment

BACKGROUND There is increasing need for better therapies to prevent the development of heart failure after myocardial infarction (MI). An injectable hydrogel derived from decellularized porcine ventricular myocardium has been shown to halt the post-infarction progression of negative left ventricular remodeling and decline in cardiac function in both small and large animal models. OBJECTIVES This study sought to elucidate the tissue-level mechanisms underlying the therapeutic benefits of myocardial matrix injection. METHODS Myocardial matrix or saline was injected into infarcted myocardium 1 week after ischemia-reperfusion in Sprague-Dawley rats. Cardiac function was evaluated by magnetic resonance imaging and hemodynamic measurements at 5 weeks after injection. Whole transcriptome microarrays were performed on RNA isolated from the infarct at 3 days and 1 week after injection. Quantitative polymerase chain reaction and histologic quantification confirmed expression of key genes and their activation in altered pathways. RESULTS Principal component analysis of the transcriptomes showed that samples collected from myocardial matrix-injected infarcts are distinct and cluster separately from saline-injected control subjects. Pathway analysis indicated that these differences are due to changes in several tissue processes that may contribute to improved cardiac healing after MI. Matrix-injected infarcted myocardium exhibits an altered inflammatory response, reduced cardiomyocyte apoptosis, enhanced infarct neovascularization, diminished cardiac hypertrophy and fibrosis, altered metabolic enzyme expression, increased cardiac transcription factor expression, and progenitor cell recruitment, along with improvements in global cardiac function and hemodynamics. CONCLUSIONS These results indicate that the myocardial matrix alters several key pathways after MI creating a pro-regenerative environment, further demonstrating its promise as a potential post-MI therapy.

Cardiospheres and tissue engineering for myocardial regeneration: potential for clinical application

•  Introduction •  Lessons from cell therapy •  Cardiac tissue engineering ‐  In vivo CTE applications ‐  In vitro CTE applications •  Conclusions

Human versus porcine tissue sourcing for an injectable myocardial matrix hydrogel

Heart failure (HF) after myocardial infarction (MI) is a leading cause of death in the western world with a critical need for new therapies. A previously developed injectable hydrogel derived from porcine myocardial matrix (PMM) has had successful results in both small and large animal MI models. In this study, we sought to evaluate the impact of tissue source on this biomaterial, specifically comparing porcine and human myocardium sources. We first developed an analogous hydrogel derived from human myocardial matrix (HMM). The biochemical and physical properties of the PMM and HMM hydrogels were then characterized, including residual dsDNA, protein content, sulfated glycosaminoglycan (sGAG) content, complex viscosity, storage and loss moduli, and nano-scale topography. Biochemical activity was investigated with in vitro studies for the proliferation of vascular cells and differentiation of human cardiomyocyte progenitor cells (hCMPCs). Next, in vivo gelation and material spread were confirmed for both PMM and HMM after intramyocardial injection. After extensive comparison, the matrices were found to be similar, yet did show some differences. Because of the rarity of collecting healthy human hearts, the increased difficulty in processing the human tissue, shifts in ECM composition due to aging, and significant patient-to-patient variability, these studies suggest that the HMM is not a viable option as a scalable product for the clinic; however, the HMM has potential as a tool for in vitro cell culture.

Cardiac cell therapy: the next (re)generation.

Heart failure remains one of the main causes of morbidity and mortality in the Western world. Current therapies for myocardial infarction are mostly aimed at blocking the progression of the disease, preventing detrimental cardiac remodeling and potentiating the function of the surviving tissue. In the last decade, great interest has arisen from the possibility to regenerate lost tissue by using cells as a therapeutic tool. Different cell types have been tested in animal models, including bone marrow-derived cells, myoblasts, endogenous cardiac stem cells, embryonic cells and induced pluripotent stem cells. After the conflicting and often inconsistent results of the first clinical trials, a step backward needs to be performed, to understand the basic biological mechanisms underlying spontaneous and induced cardiac regeneration. Current studies aim at finding new strategies to enhance cellular homing, survival and differentiation in order to improve the overall outcome of cellular cardiomyoplasty

New Perspectives to Repair a Broken Heart

The aim of cardiac cell therapy is to restore at least in part the functionality of the diseased or injured myocardium by the use of stem/progenitor cells. Recent clinical trials have shown the safety of cardiac cell therapy and encouraging efficacy results. A surprisingly wide range of non-myogenic cell types improves ventricular function, suggesting that benefits may result in part from mechanisms that are distinct from true myocardial regeneration. While clinical trials explore cells derived from skeletal muscle and bone marrow, basic researchers are investigating sources of new cardiomyogenic cells, such as resident myocardial progenitors and embryonic stem cells. In this commentary we briefly review the evolution of cell-based cardiac repair, some progress that has been made toward this goal, and future perspectives in the regeneration of cardiac tissue.