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Background Mesenchymal stromal cells (MSC) repair infarcted hearts mainly through paracrine mechanisms. Low cell engraftment limits the release of soluble paracrine factors (SF) over time and, consequently, MSC efficacy. We tested whether a synthetic extracellular matrix mimic, a hydrogel containing heparin (H-HG), could ameliorate MSC engraftment and binding/release of SF, thus improving MSC therapy efficacy. Methods and results In vitro, rat bone-marrow MSC (rBM-MSC) were seeded and grown into H-HG. Under normoxia, the hydrogel did not affect cell survival (rBM-MSC survival >90% at each time point tested); vice versa, under hypoxia the biomaterial resulted to be protective for the cells (p H-HG or control PEG hydrogels (HG) were incubated with VEGF or bFGF for binding/release quantification. Data showed significantly higher amount of VEGF and bFGF bound by H-HG compared with HG (p In vivo, myocardial infarction (MI) was induced in female Sprague Dawley rats by permanent coronary ligation. One week later, saline, rBM-MSC, H-HG or rBM-MSC/H-HG were injected in the infarct zone. The co-injection of rBM-MSC/H-HG into infarcted hearts significantly increased cardiac function. Importantly, we observed a significant gain in MSC engraftment, reduction of ventricular remodeling and stimulation of neo-vasculogenesis. We also documented higher amounts of several pro-angiogenic factors in hearts treated with rBM-MSC/H-HG. Conclusions Our data show that H-HG increases MSC engraftment, efficiently fine tunes the paracrine MSC actions and improves cardiac function in infarcted rat hearts. Statement of Significance Transplantation of MSC is a promising treatment for ischemic heart disease, but low cell engraftment has so far limited its efficacy. The enzymatically degradable H-HG that we developed is able to increase MSC retention/engraftment and, at the same time, to fine-tune the paracrine effects mediated by the cells. Most importantly, the co-transplantation of MSC and H-HG in a rat model of ischemic cardiomyopathy improved heart function through a significant reduction in ventricular remodeling/scarring and amelioration in neo-vasculogenesis/endogenous cardiac regeneration. These beneficial effects are comparable to those obtained by others using a much greater number of cells, strengthening the efficacy of the biomaterial used in increasing the therapeutic effects of MSC. Given its efficacy and safety, documented by the absence of immunoreaction, our strategy appears readily translatable to clinical scenarios.

The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC-CM by cytokine array. Our results strongly support the concept that the administration of hAMC-CM favors the repair process after acute myocardial infarction. Significance The demonstration that stem cells repair infarcted hearts mainly through paracrine mechanisms represents a potential breakthrough. Characterization of therapeutic paracrine mediators could lead to the possibility of treating acute myocardial infarction (AMI) with a single stem cell-derived molecule or a mixture. Compared with cell therapy, this approach would be technically easier to translate to the bedside. An even more straightforward strategy consists of the administration of the entire stem cell secretome (i.e., conditioned medium [CM]). Despite these potential advantages, this approach has not been thoroughly investigated using human cells. This study shows that CM of fetal stromal cells (human amniotic membrane-derived mesenchymal stromal cell [hAMC]-CM), derived from an ethically acceptable source such as the placenta, can repair infarcted hearts without the need for any manipulation. The use of hAMC-CM might be readily translated to the clinical arena in the setting of AMI upon demonstration of its effectiveness in a large animal model.

Mesenchymal stem cells (MSC) possess high plasticity and the potential to differentiate into several different cell types; this characteristic has implications for cell therapy and reparative biotechnologies. MSC have been originally isolated from the bone marrow (BM-MSC), but they have been found also in other tissues such as adipose tissue, cord blood, synovium, skeletal muscle, and lung. MSC are able to differentiate in vitro and in vivo into several cell types such as bone, osteocytes, chondrocytes, adipocytes, and skeletal myocytes, just to name a few.During the last two decades, an increasing number of studies have proven the therapeutic potential of MSC for the treatment of neurodegenerative diseases, spinal cord and brain injuries, cardiovascular diseases, diabetes mellitus, and diseases of the skeleton. Their immuno-privileged profile allows both autologous and allogeneic use. For all these reasons, the scientific appeal of MSC is constantly on the rise.The identity of MSC is currently based on three main criteria: plastic-adherence capacity, defined epitope profile, and capacity to differentiate in vitro into osteocytes, chondrocytes, and adipocytes. Here, we describe standard protocols for the differentiation of BM-MSC into the osteogenic, chondrogenic, and adipogenic lineages.

Tissue regeneration from transplanted mesenchymal stromal cells (MSC) either through transdifferentiation or cell fusion was originally proposed as the principal mechanism underlying their therapeutic action. However, several studies have now shown that both these mechanisms are very inefficient. The low MSC engraftment rate documented in injured areas also refutes the hypothesis that MSC repair tissue damage by replacing cell loss with newly differentiated cells. Indeed, despite evidence of preferential homing of MSC to the site of myocardial ischemia, exogenously administered MSC show poor survival and do not persist in the infarcted area. Therefore, it has been proposed that the functional benefits observed after MSC transplantation in experimental models of tissue injury might be related to the secretion of soluble factors acting in a paracrine fashion. This hypothesis is supported by pre-clinical studies demonstrating equal or even improved organ function upon infusion of MSC-derived conditioned medium (MSC-CM) compared with MSC transplantation. Identifying key MSC-secreted factors and their functional role seems a reasonable approach for a rational design of nextgeneration MSC-based therapeutics. Here, we summarize the major findings regarding both different MSC-mediated paracrine actions and the identification of paracrine mediators.

Mesenchymal stem cells (MSC) produce and secrete a great variety of cytokines and chemokines that play beneficial paracrine actions when MSC are used for tissue repair. The conditioned medium (CM) derived from MSC can be used both in vitro and in vivo to test specific paracrine effects or to screen putative paracrine/autocrine mediators by proteomics.In this chapter, we describe a straightforward method to prepare MSC-derived CM. Furthermore, we summarize some in vitro assays useful for testing the cytoprotective, angiogenic, and regenerative activity of CM. These assays are very helpful when studying the role of MSC in cardiac repair and regeneration.

Mesenchymal stem cells (MSC) produce and secrete a great variety of cytokines and chemokines that play beneficial paracrine actions when MSC are used for tissue repair. The conditioned medium (CM) derived from MSC can be used both in vitro and in vivo to test specific paracrine effects or to screen putative paracrine/autocrine mediators by proteomics.In this chapter, we describe a straightforward method to prepare MSC-derived CM. Furthermore, we summarize some in vitro assays useful for testing the cytoprotective, angiogenic, and regenerative activity of CM. These assays are very helpful when studying the role of MSC in cardiac repair and regeneration.

Tissue regeneration from transplanted mesenchymal stromal cells (MSC) either through transdifferentiation or cell fusion was originally proposed as the principal mechanism underlying their therapeutic action. However, several studies have now shown that both these mechanisms are very inefficient. The low MSC engraftment rate documented in injured areas also refutes the hypothesis that MSC repair tissue damage by replacing cell loss with newly differentiated cells. Indeed, despite evidence of preferential homing of MSC to the site of myocardial ischemia, exogenously administered MSC show poor survival and do not persist in the infarcted area. Therefore, it has been proposed that the functional benefits observed after MSC transplantation in experimental models of tissue injury might be related to the secretion of soluble factors acting in a paracrine fashion. This hypothesis is supported by pre-clinical studies demonstrating equal or even improved organ function upon infusion of MSC-derived conditioned medium (MSC-CM) compared with MSC transplantation. Identifying key MSC-secreted factors and their functional role seems a reasonable approach for a rational design of nextgeneration MSC-based therapeutics. Here, we summarize the major findings regarding both different MSC-mediated paracrine actions and the identification of paracrine mediators.

In immunoglobulin (Ig) light-chain (LC) (AL) amyloidosis, AL deposition translates into life-threatening cardiomyopathy. Clinical and experimental evidence indicates that soluble cardiotoxic LCs are themselves harmful for cells, by which they are internalized. Hypothesizing that interaction of soluble cardiotoxic LCs with cellular proteins contributes to damage, we characterized their interactome in cardiac cells. LCs were purified from patients with AL amyloidosis cardiomyopathy or multiple myeloma without amyloidosis (the nonamyloidogenic/noncardiotoxic LCs served as controls) and employed at concentrations in the range observed in AL patients' sera. A functional proteomic approach, based on direct and inverse coimmunoprecipitation and mass spectrometry, allowed identifying LC-protein complexes. Findings were validated by colocalization, fluorescence lifetime imaging microscopy (FLIM)-fluorescence resonance energy transfer (FRET), and ultrastructural studies, using human primary cardiac fibroblasts (hCFs) and stem cell-derived cardiomyocytes. Amyloidogenic cardiotoxic LCs interact in vitro with specific intracellular proteins involved in viability and metabolism. Imaging confirmed that, especially in hCFs, cardiotoxic LCs (not controls) colocalize with mitochondria and spatially associate with selected interactors: mitochondrial optic atrophy 1-like protein and peroxisomal acyl-coenzyme A oxidase 1 (FLIM-FRET efficiencies 11 and 6%, respectively). Cardiotoxic LC-treated hCFs display mitochondrial ultrastructural changes, supporting mitochondrial involvement. We show that cardiotoxic LCs establish nonphysiologic protein-protein contacts in human cardiac cells, offering new clues on the pathogenesis of AL cardiomyopathy.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. Stem Cells 2015;33:1187–1199

Purpose: The in vivo effects of bone marrow mesenchymal stem cells (BM-MSC) in repairing ischemic hearts are limited by poor engraftment, low differentiation rate and, in elderly patients, by reduced paracrine properties. To overcome these limitations, we genetically engineered BM-MSC with a novel bicistronic lentivirus co-expressing IGF1 and BMP2 (IB), two factors involved in both cardiac differentiation and cytoprotection. Methods: We cloned an IRES-based expression cassette into a novel lentiviral vector. Rat BM-MSC were transduced with a control (GFP-MSC) or IB (IB-MSC) virus. Cardiac differentiation was evaluated by RT-qPCR, western blot, and immunocytochemistry for cardiac specific markers such as GATA4, Nkx2.5, Tbx5, Mef2c, Cx43, cTnT, a-MHC, a-SA and a-SR1. Resistance to hypoxia-induced apoptosis was measured after 24hrs of hypoxia. To study the cytoprotective paracrine properties, H9c2 cells were deprived of oxygen and glucose (6hrs), and then reoxygenated (18hrs) in the presence of control medium (CTRL-M) or medium conditioned by either GFP-MSC (GFP-CM) or IB-MSC (IB-CM). Viability was measured by MTS assay, apoptosis by caspase-3 activation, cytotoxicity by LDH release. Transcriptional levels of known pro- and anti-apoptotic genes were measured by RT-PCR. Activation of I and B associated pathways (Akt, ERK1/2, and SMAD1/5/8) was assessed by western blot. Results: The transduction efficiency with our novel vector was extremely high (>90% with one cycle of infection). Compared with GFP-MSC, IB-MSC triggered expression of cardiac markers both at RNA (Tbx5 +3.8, p

Purpose: It has been demonstrated that miR1 and miR133 are involved in cardiac development and stem cell differentiation, while miR499 enhances the differentiation of embryonic and adult stem cells into cardiomyocytes (CMC). However, it is currently unknown if these miRNA may act synergistically to improve differentiation efficiency. Methods: Mouse P19 cells transduced with a viral vector expressing GFP under the control of a cTnI promoter were primed with 0.5% of DMSO and used as cardiogenic differentiation model (P19 CTRL). MiR499 was transiently over-expressed in P19 cells individually or together with miR1 or miR133. Moreover, we differentiated P19 cells with pre-miR precursors without DMSO. Cardiac differentiation was quantified by: 1) count of spontaneous beating areas; 2) qPCR of the cardiac markers Nkx2.5, GATA4, MEF2c, MLC2v, Cx43 and cTnT; 3) Western Blot and ICC of cTnT and Cx43; 4) measure of inducibility of RyR-mediated Ca++ transients in P19 exposed to caffeine; 5) recording of mechanical activity after Ca++ depletion and in the presence of nifedipine or ryanodine. Results: The overexpression of miR499 alone increased the number of beating CMC by 1.7 fold compared with P19 CTRL (p

Purpose: Mesenchymal stem cells (MSC) derived form human amniotic membrane (A-MSC) differentiate into cardiomyocyte (CMC) more promptly than bone marrow-derived MSC (BM-MSC). Still, the differentiation efficiency is not very high. We found that BM-MSC, particularly from elderly patients, express lower amount of several proteins potentially involved in cardiac differentiation compared with A-MSC. We hypothesized that by overexpressing these factors, cardiac differentiation efficiency of A-MSC may further increase. Methods: Human A-MSC were transduced with a lentiviral vector encoding for either GFP alone (GFP-A-MSC control cells), or TGFβ1 (TGFβ1-A-MSC), or BMP2 (BMP2-A-MSC), or IGF-I (IGF-I-A-MSC), or FGF2 (FGF2-A-MSC). To confirm transduction efficiency, after 48 hours the cells were analyzed by FACS, Western Blot (WB), ELISA and immunocytochemistry (ICC). The cells were then maintained in culture for 5 weeks and analyzed by qPCR, WB and ICC at two different time points: 2 weeks, to search for expression of early cardiac genes (GATA4, Nkx2.5 and Tbx5), and 5 weeks, to verify acquisition of cardiac-specific proteins (Cx43, cTnT and α–MHC). Results: The transduction efficiency with all four lentiviral vectors was higher than 90%. WB, ELISA and ICC confirmed the over-expression of the protein of interest. Compared with GFP-A-MSC, the over-expression of each factor triggered the expression of cardiac markers. qPCR and ICC showed the over-expression of GATA4 and Nkx2.5 in IGF-I-A-MSC and of Tbx5 in TGFβ1-A-MSC and FGF2-A-MSC. qPCR also documented that IGF-I and FGF2 significantly increased the expression levels of Cx43 (+15.8 and +9.5 vs GFP-A-MSC respectively, p