Claudia Serradifalco

Embryonic and foetal Islet-1 positive cells in human hearts are also positive to c-Kit

During embryogenesis, the mammalian heart develops from a primitive heart tube originating from two bilateral primary heart fields located in the lateral plate mesoderm. Cells belongings to the pre-cardiac mesoderm will differentiate into early cardiac progenitors, which express early transcription factors which are also common to the Isl-1 positive cardiac progenitor cells isolated from the developing pharyngeal mesoderm and the foetal and post-natal mice hearts. A second population of cardiac progenitor cells positive to c-Kit has been abundantly isolated from adult hearts. Until now, these two populations have been considered two different sets of progenitor cells present in the heart in different stages of an individual life. In the present study we collected embryonic, foetal and infant hearts, and we tested the hypotheses that c-Kit positive cells, usually isolated from the adult heart, are also present in the intra-uterine life and persist in the adult heart after birth, and that foetal Isl-1 positive cells are also positive to c-Kit. Using immunohistochemistry we studied the temporal distribution of Isl-1 positive and c-Kit/CD105 double positive cells, and by immunofluorescence and confocal analysis we studied the co-localization of c-Kit and Isl-1 positive cells. The results indicated that cardiomyocytes and interstitial cells were positive for c-Kit from the 9th to the 19h gestational week, that cells positive for both c-Kit and CD105 appeared in the interstitium at the 17h gestational week and persisted in the postnatal age, and that the Isl-1 positive cells were a subset of the c-Kit positive population.

Silk fibroin scaffolds enhance cell commitment of adult rat cardiac progenitor cells

The use of three‐dimensional (3D) cultures may induce cardiac progenitor cells to synthesize their own extracellular matrix (ECM) and sarcomeric proteins to initiate cardiac differentiation. 3D cultures grown on synthetic scaffolds may favour the implantation and survival of stem cells for cell therapy when pharmacological therapies are not efficient in curing cardiovascular diseases and when organ transplantation remains the only treatment able to rescue the patients life. Silk fibroin‐based scaffolds may be used to increase cell affinity to biomaterials and may be chemically modified to improve cell adhesion. In the present study, porous, partially orientated and electrospun nanometric nets were used. Cardiac progenitor cells isolated from adult rats were seeded by capillarity in the 3D structures and cultured inside inserts for 21 days. Under this condition, the cells expressed a high level of sarcomeric and cardiac proteins and synthesized a great quantity of ECM. In particular, partially orientated scaffolds induced the synthesis of titin, which is a fundamental protein in sarcomere assembly. Copyright

MicroRNA and Cardiac Stem Cell Therapy

Cardiac Progenitor Cells (CPCs) are multipotent cells of the myocardium. They are located inside niches of the heart muscle, can be isolated, characterized and used for cardiac regeneration in stem cell therapy. Actually, CPCs may be isolated by tissue digestion with or without cell sorting, but it is difficult to achieve the maximum level of differentiation when these cells are implanted into a damaged myocardium. The knowledge recently acquired on small molecules of non-coding RNAs, microRNA (miRNA), may improve the use of these cells in stem cell therapy. In fact, these small molecules may be attached to devices or adminstered as they are or in combination with nanoparticles in order to drive the correct differentiation of stem cells. Regarding heart regeneration, we can acquire knowledge from the role of miRNAs in heart development and use it to reprogram CPCs to gain the correct three-dimensional structure of the cardiac muscle.

Adult stem cells, scaffolds for in vivo and in vitro myocardial tissue engineering

The main goal in the last few years in cardiac research has been to isolate cardiac potential stem cells from adult myocardium and to demonstrate their differentiation potential. We have previously demonstrated that c-Kit positive cardiac stem cells are able to organize themselves into a tissue-like cell mass. In this 3D mass, they can produce a high concentration of natural extracellular matrix, can create vessels, a capsule and, with the help of an Open-pore Polylactic Acid scaffold, many cells can organize an elementary myocardium. Drawing from this background, we decided to design and use poly-lactic scaffolds and the model of the athymic Nude-Foxn1(nu) mouse to evaluate the extent of the myogenic vs endothelial differentiation in vivo, and to evaluate the presence or the absence of a foreign body reaction.

Rat Cardiac progenitor cells and their application in cell therapy

cells and the subsequent heart failure. When the pharmacological approach no longer complies with the disease evolution, organ transplantation appears to be the only treatment able to rescue the patient life. Cell therapy promises to be clinically efficient and would allow circumventing many limitations of organ transplantation, such as organ low availability, major surgical procedures, high costs and longterm immunosuppression [1]. We designed porous Poly-Lactic Acid (PLLA) and Fibroin scaffolds to deliver CPCs in the heart, we isolated and characterized CPCs for the expression of c-Kit, MDR-1 and Sca-1 by flow cytometry, we tested their degree of differentiation in vitro studying the expression of all known rat sarcomeric proteins by real-time PCR and their differentiated morphology on Electron Microscopy samples [2]. A particular attention was given to the expression of microRNA, because their role in the differentiation process of cardiac precursors is emerging. We also tested the host reaction to scaffolds, CPCs, and CPCs/scaffolds. In vivo, almost all the used scaffolds induced a foreign body reaction in nude mouse and rats, but not in SCID mice. Cardiac stem cells a T cell-mediated immune response induced in nude mice, letting us suppose that, differently from Mesenchymal Stem Cells, they express MHC molecules on their surface. The degree of differentiation, the expression of ECM and integrin proteins, and the expression of several sarcomeric proteins were dependent on the type of scaffold and the polymer used.

Poly-lactic acid and fibrinoin scaffolds as three-dimensional device to differentiate cardiac stem cells: in vitro and in vivo studies

Introduction The rapid translation of preclinical cell-based therapy to restore damaged myocardium has raised questions concerning the best cell type as well as the best delivery route, and the best time of cell injection into the myocardium. Intramyocardial injection of stem cells is by far the most-used delivery technique in preclinical studies. We have recently demonstrated that c-Kit+ cardiac progenitor cells are able to organize themselves into a tissue-like cell mass in three-dimensional cultures, and with the help of an OPLA scaffold, many cells can create an organized elementary myocardium. Hypothesis We designed random porosity and oriented poly-lactic acid and fibrinoin scaffolds to be used as three-dimensional systems to favour differentiation of cardiac progenitor cells. We tested them in vitro and in vivo to study their potentiality as surgical devices in cardiology. Methods For the synthesis of PDLLA scaffolds, the Poly (D,L lactic acid) (RESOMER® 207, MW = 252 kDa) polimer were used (6.7%) in Dicloromethane/Dimetilformamide (DCM/DMF) 70/30 (v/v). The three-dimensional structure was obtained by salt-leaching, using NaCl crystals as porosity agent (NaCl < 224 μm and <150 μm). For the synthesis of fibrinoin scaffolds, degummed silk fibres were dried and dissolved into 9.3 m LiBr water solution (20% w/v) at 65°C for 3h. Scaffolds with different porosities, pore size, and properties were made by freeze-drying and salt-leaching. Scaffolds embedded with collagen I and cardiac progenitor cells were implanted in the subcutaneous dorsal region of athymic Nude-Foxn1nu mice. Results Cardiac progenitor cells differentiated into cardiomyocytes in vitro into PDLLA scaffolds in a M-199 medium supplemented with 20% FBS within 21 days. A less extent of differentiation has been obtained in the dorsal subcutaneous region, and a foreign body reaction has been recorded.