Elvira Forte

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.

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.

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

TGFβ-Dependent Epithelial-to-Mesenchymal Transition Is Required to Generate Cardiospheres from Human Adult Heart Biopsies

Autologous cardiac progenitor cells (CPCs) isolated as cardiospheres (CSps) represent a promising candidate for cardiac regenerative therapy. A better understanding of the origin and mechanisms underlying human CSps formation and maturation is undoubtedly required to enhance their cardiomyogenic potential. Epithelial-to-mesenchymal transition (EMT) is a key morphogenetic process that is implicated in the acquisition of stem cell-like properties in different adult tissues, and it is activated in the epicardium after ischemic injury to the heart. We investigated whether EMT is involved in the formation and differentiation of human CSps, revealing that an up-regulation of the expression of EMT-related genes accompanies CSps formation that is relative to primary explant-derived cells and CSp-derived cells grown in a monolayer. EMT and CSps formation is enhanced in the presence of transforming growth factor β1 (TGFβ1) and drastically blocked by the type I TGFβ-receptor inhibitor SB431452, indicating that TGFβ-dependent EMT is essential for the formation of these niche-like 3D-multicellular clusters. Since TGFβ is activated in the myocardium in response to injury, our data suggest that CSps formation mimics an adaptive mechanism that could potentially be enhanced to increase in vivo or ex vivo regenerative potential of adult CPCs.

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.

Different types of cultured human adult cardiac progenitor cells have a high degree of transcriptome similarity.

The discovery and isolation of different resident cardiac progenitor cells (CPCs) a decade ago, as described by several research groups, stimulated the use of these cells for cardiac regeneration. Human CPCs are moving towards the clinic as one of the most promising cell types for cardiac repair, but the extent to which their molecular profiles vary as a result of donor heterogeneity or different isolation methods remain unclear. Defining a common molecular profile that defines CPC’s is therefore an important goal. Similarly, identifying robust and multilaboratory isolation and culture protocols that generate reproducible cell populations from genetically diverse donors is critical for their translational success. In this respect, we collected human auricle biopsy samples anonymously from 20 different adult patients that underwent bypass surgery and generated a total number of 33 different cardiac derived progenitor cell (CPC) lines (Table S1). Human CPCs were isolated according the original published protocol, based on c-kit 1 or Sca-1 2 expression or auricles were cut in 1 mm3 parts and cultured as explants to obtain Cardiospheres (CSps) 3 and Cardiosphere Derived Cells (CDCs) 4. CPCs were subsequently propagated in a panel of different media formulations, either in their originally described culture media or switched to media and culture coatings of the other CPC subsets (Figure S1, Table S7). When comparing individual CPC cell-lines, isolated with different methodologies, they shared a high degree of similarities and correlation in gene expression patterns (Fig. ​(Fig.1B).1B). By averaging expression profiles of individual CPC conditions, thereby reducing donor variability, similarities increased even more, ranging from 0.92 to 0.96 (Fig. ​(Fig.1C;1C; Table S2). These results suggest that individual donor differences were larger than influences of isolation and medium conditions. Moreover, the strongest correlations between the different CPC lines were observed when cells were isolated and cultured in the same conditions. Among the different CPCs, spheres-growing CSps showed the least correlation (0.91–0.96), while monolayer-growing CPCs shared higher correlations among them (0.96–0.98). We performed a moderated t-test to evaluate significant differentially expressed genes between the individual samples (Tables S3 and S4). Out of the 13,073 analysd genes, we found only few genes differentially expressed in 5 of 20 different monolayer-cultured CPC cell-lines comparisons. Only when the 3D-cultured CSps were compared with the other CPCs more differently expressed genes could be identified. Although only limited genes were different, we further explored if we could identify differences in gene patterns between the different CPC populations, based on selected genes important for stem cell-maintenance, their growth and biology. In particular, we evaluated genes involved in the regulation of different stem cell pathways like TGF-β, Wnt, NFkB, p53, JAK/STAT, Notch and Hedgehog (Fig. S2A), cell cycle (Fig. S2B), stem cell transcription factors (Fig. S2C), and growth factors, cytokines and chemokines (Fig. S2D). Detailed heat map analysis showed again; however, a very similar profile among all samples, with small differences mainly related to individual donors and not to different cell types or conditions (Fig. S2). Since CSps and monolayer growing CPCs have differently expressed patterns, we selected all the significantly differentially expressed genes that displayed a two fold or more difference and compared them with CDCs, and c-Kit and Sca-1+ CPCs monolayer-cultures (Table S6). Ingenuity pathway analysis identified a gene network in CSps that is enriched in genes encoding for growth factor production and signalling molecules involved in the development of cardiac muscle, vasculogenesis and angiogenesis (Fig. ​(Fig.2).2). Among them BMP-2, HGF, LIF, PTGS-2, VEGFA and PDGFRB are known to play an important role during cardiac development. Moreover, having a protective effect on a developing heart failure. Figure 1 Experimental design of the project (A) and hierarchical clustering of CPCs samples (B and C). Sca-1+ cells were isolated from human auricle biopsy and cultured in gelatin coated flask and Sca-1 medium (Sca GEL S-MED) (2) (d). After expansion cells were ... Figure 2 Ingenuity molecular networks analysis of the differentially expressed genes. Fold difference ≥2; p<0.05. (A) Differentially regulated genes between CSps and Sca GEL SP++ in Cardiovascular System Development and Function, Embryonic Development, ... Taken together, our data suggest that human CPCs can be isolated from patient heart biopsies using different markers, such as c-kit or Sca-1- like, and alternative methodologies, via direct cell isolation or via explant culture, such as CSps and CDCs. For the first time, however, we showed that upon culture expansion, these cell populations have a very similar gene expression profile, even more pronounced when cultured in comparable culture conditions and even transcended by donor differences. Among the different CPCs analysed, CSps are the most different, probably because of the unselected cell populations and containing more supporting cell population that form CSps and their particular 3D culture structure and thereby different interactions and growing conditions. Surprisingly CDCs, which is a cell population derived from CSps, are more similar with other antigen selected CPCs rather than with CSps, confirming the idea that monolayer and high proliferative culture condition might play an important role in minimizing the differences among the different CPCs analysed. Recently, Dey et al. isolated murine CPCs, based on different surface markers 5, and showed that these, non-cultured cells, represent progenitor cell populations at different stages of cardiac commitment 5. In our study, we did not observe such differences between the different human monolayer CPCs population upon culture propagation. A similar stage difference, however, might be present in situ in humans as well but lost upon culture expansion. The expression of these different stem cell markers and their co-expression probably represent different developmental and/or physiological stages of CPCs, rather than intrinsic different CPC populations. For future translation for cardiac cell therapy, our results suggest that we need to take into account the cell donor variability between patients more than the isolation methodology, and further study the correlation between CPC characteristics and e.g. the diseased status of a patient. Our findings are of fundamental importance to create a consensus among different scientists in the field of myocardial regeneration, which should help align future clinical approaches to improve the reported beneficial effects of cell therapy for heart disease by using cardiac derived progenitor cell populations.

Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart

Experimental data suggest that cell‐based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit+ CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild‐type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate ‘cardiospheres’, a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit+ CSCs.

From ontogenesis to regeneration: Learning how to instruct adult cardiac progenitor cells

Since the first observations over two centuries ago by Lazzaro Spallanzani on the extraordinary regenerative capacity of urodeles, many attempts have been made to understand the reasons why such ability has been largely lost in metazoa and whether or how it can be restored, even partially. In this context, important clues can be derived from the systematic analysis of the relevant distinctions among species and of the pathways involved in embryonic development, which might be induced and/or recapitulated in adult tissues. This chapter provides an overview on regeneration and its mechanisms, starting with the lesson learned from lower vertebrates, and will then focus on recent advancements and novel insights concerning regeneration in the adult mammalian heart, including the discovery of resident cardiac progenitor cells (CPCs). Subsequently, it explores all the important pathways involved in regulating differentiation during development and embryogenesis, and that might potentially provide important clues on how to activate and/or modulate regenerative processes in the adult myocardium, including the potential activation of endogenous CPCs. Furthermore the importance of the stem cell niche is discussed, and how it is possible to create in vitro a microenvironment and culture system to provide adult CPCs with the ideal conditions promoting their regenerative ability. Finally, the state of clinical translation of cardiac cell therapy is presented. Overall, this chapter provides a new perspective on how to approach cardiac regeneration, taking advantage of important lessons from development and optimizing biotechnological tools to obtain the ideal conditions for cell-based cardiac regenerative therapy.