Ex vivo expansion in a clinical grade medium, containing growth factors from human platelets, enhances migration capacity of adipose stem cells.

Introduction: Adipose tissue mesenchymal stem/stromal cells (ASC) can be used as advanced therapy medicinal product in regenerative and cancer medicine. We previously demonstrated Supernatant Rich in Growth Factors (SRGF) can replace fetal bovine serum (FBS) to expand ASC by a clinical grade compliant protocol. The therapeutic potential of ASC is based also on their homing capacity toward inflammatory/cancer sites: oriented cell migration is a fundamental process in this scenario. We investigated the impact of SRGF on ASC migration properties. Methods: The motility/migration potential of ASC expanded in 5% SRGF was analyzed, in comparison to 10% FBS, by standard wound healing, bidimensional chemotaxis and transwell assays, and by millifluidic transwell tests. Mechanisms involved in the migration process were investigated by transient protein overexpression. Results: In comparison to standard 10% FBS, supplementation of the cell culture medium with 5% SRGF, strongly increased migration properties of ASC along the chemotactic gradient and toward cancer cell derived soluble factors, both in static and millifluidic conditions. We showed that, independently from applied migratory stimulus, SRGF expanded ASC were characterized by far lower expression of α-smooth muscle actin (αSMA), a protein involved in the cell migration machinery. Overexpression of αSMA induced a significant and marked decrease in migration capacity of SRGF expanded ASC. Discussion: In conclusion, 5% SRGF addition in the cell culture medium increases the migration potential of ASC, reasonably through appropriate downregulation of αSMA. Thus, SRGF could potentially improve the therapeutic impact of ASC, both as modulators of the immune microenviroment or as targeted drug delivery vehicles in oncology.

IRE1a-Induced FilaminA Phosphorylation Enhances Migration of Mesenchymal Stem Cells Derived from Multiple Myeloma Patients.

Multiple myeloma (MM) is an aggressive malignancy that shapes, during its progression, a pro-tumor microenvironment characterized by altered protein secretion and the gene expression of mesenchymal stem cells (MSCs). In turn, MSCs from MM patients can exert an high pro-tumor activity and play a strong immunosuppressive role. Here, we show, for the first time, greater cell mobility paralleled by the activation of FilaminA (FLNA) in MM-derived MSCs, when compared to healthy donor (HD)-derived MSCs. Moreover, we suggest the possible involvement of the IRE1a-FLNA axis in the control of the MSC migration process. In this way, IRE1a can be considered as a good target candidate for MM therapy, considering its pro-survival, pro-osteoclast and chemoresistance role in the MM microenvironment. Our results suggest that IRE1a downregulation could also interfere with the response of MSCs to MM stimuli, possibly preventing cell-cell adhesion-mediated drug resistance. In addition, further investigations harnessing IRE1a-FLNA interaction could improve the homing efficiency of MSC as cell product for advanced therapy applications.

Modified mesenchymal stem cells in cancer therapy: A smart weapon requiring upgrades for wider clinical applications.

Mesenchymal stem stromal cells (MSC) are characterized by the intriguing capacity to home toward cancer cells after systemic administration. Thus, MSC can be harnessed as targeted delivery vehicles of cytotoxic agents against tumors. In cancer patients, MSC based advanced cellular therapies were shown to be safe but their clinical efficacy was limited. Indeed, the amount of systemically infused MSC actually homing to human cancer masses is insufficient to reduce tumor growth. Moreover, induction of an unequivocal anticancer cytotoxic phenotype in expanded MSC is necessary to achieve significant therapeutic efficacy. Ex vivo cell modifications are, thus, required to improve anti-cancer properties of MSC. MSC based cellular therapy products must be handled in compliance with good manufacturing practice (GMP) guidelines. In the present review we include MSC-improving manipulation approaches that, even though actually tested at preclinical level, could be compatible with GMP guidelines. In particular, we describe possible approaches to improve MSC homing on cancer, including genetic engineering, membrane modification and cytokine priming. Similarly, we discuss appropriate modalities aimed at inducing a marked cytotoxic phenotype in expanded MSC by direct chemotherapeutic drug loading or by genetic methods. In conclusion, we suggest that, to configure MSC as a powerful weapon against cancer, combinations of clinical grade compatible modification protocols that are currently selected, should be introduced in the final product. Highly standardized cancer clinical trials are required to test the efficacy of ameliorated MSC based cell therapies.

miR-335-laden B Cell-Derived Extracellular Vesicles Promote SOX4-Dependent Apoptosis in Human Multiple Myeloma Cells.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow. Despite novel therapies, MM still remains an incurable cancer and new strategies are needed. Increased expression of the transcription factor Sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4) has been correlated with tumor development and progression through a variety of distinct processes, including inhibition of apoptosis, increased cell invasion and metastasis, and induction and maintenance of cancer-initiating cells. The role of SOX4 in MM is largely unknown. Since SOX4 is a known target of miR-335, we used miR-335 to assess whether SOX4 modulation could promote apoptosis in MM cells. Using an MM cell model we show that miR-335 acts both on SOX4-related genes (AKT, PI3K) and hypoxia-inducible factor 1-alpha (Hif1-α). In addition, we show miR-335-laden extracellular vesicles induced in B cells (iEVs) are also effective in targeting SOX4, causing apoptosis. Collectively, we propose that miR-335-laden iEVs could be developed as a novel form of gene therapy in MM.

Nucleofection of Adipose Mesenchymal Stem/Stromal Cells: Improved Transfection Efficiency for GMP Grade Applications.

Nucleofection (NF) is a safe, non-viral transfection method, compatible with Good Manufacturing Practice guidelines. Such a technique is useful to improve therapeutic effectiveness of adipose tissue mesenchymal stem cells (ASC) in clinical settings, but improvement of NF efficiency is mandatory. Supernatant rich in growth factors (SRGF) is a clinical-grade medium additive for ASC expansion. We showed a dramatically increased NF efficiency and post-transfection viability in ASC expanded in presence of SRGF (vs. fetal bovine serum). SRGF expanded ASC were characterized by increased vesicle endocytosis but lower phagocytosis properties. SRGF increased n-6/n-3 ratio, reduced membrane lipid raft occurrence, and lowered intracellular actin content in ASC. A statistical correlation between NF efficiency and lipid raft availability on cell membranes was shown, even though a direct relationship could not be demonstrated: attempts to selectively modulate lipid rafts levels were, in fact, limited by technical constraints. In conclusion, we reported for the first time that tuning clinical-grade compatible cell culture conditions can significantly improve ASC transfection efficiency by a non-viral and safe approach. A deep mechanistic characterization is extremely complex, but we can hypothesize that integrated changes in membrane structure and intracellular actin content could contribute to explain SRGF impact on ASC NF efficiency.

Adipose mesenchymal stromal/stem cells expanded by a GMP compatible protocol displayed improved adhesion on cancer cells in flow conditions.

BACKGROUND: Adipose tissue derived mesenchymal stromal/stem cells (ASC) can be expanded using supernatant rich in growth factors (SRGF) as Good Manufacturing Practice compatible additive, instead of fetal bovine serum (FBS). After transendothelial migration, ASC can migrate to cancer masses where they can release active substances. Due to their homing and secretion properties ASC can be used as targeted drug delivery vehicles. Nevertheless, the fraction of ASC actually reaching the tumor target is limited. The impact of culture conditions on ASC homing potential on cancer cells is unknown. METHODS: In dynamic in vitro conditions, we perfused FBS or SRGF ASC in flow chambers coated with collagen type I and fibronectin or seeded with endothelial cells or with HT1080, T98G and Huh7 cancer cells. Expression of selected adhesion molecules was evaluated by standard cytofluorimetry. Dynamic intracellular calcium concentration changes were evaluated in microfluidic and static conditions. RESULTS: When compared to FBS ASC, not specific adhesion of SRGF ASC on collagen type I and fibronectin was lower (-33.9%±12.2% and -45.3%±16.9%), while on-target binding on HT1080 and T98G was enhanced (+147%±8% and 120.5%±5.2%). Adhesion of both FBS and SRGF ASC on Huh7 cells was negligible. As confirmed by citofluorimetry and by function-blocking antibody, SRGF mediated decrease of CD49a expression accounted for lower SRGF-ASC avidity for matrix proteins. Upon stimulation with calcium ionophore in static conditions, mobilization of intracellular calcium in SRGF ASC was greater than in FBS ASC. In dynamic conditions, upon adhesion on matrix proteins and HT1080 cells, SRGF ASC showed marked oscillatory calcium concentration changes. CONCLUSIONS: SRGF can enhance specific ASC binding capacity on selected cancer cells as HT1080 (fibrosarcoma) and T98G (glioblastoma) cells. Upon cell-cell adhesion, SRGF ASC activate intracellular responses potentially improving cell secretion functions. SRGF ASC could be considered as suitable drug delivery vehicle for cancer therapy.

Atrial myxomas arise from multipotent cardiac stem cells.

AIMS: Cardiac myxomas usually develop in the atria and consist of an acid-mucopolysaccharide-rich myxoid matrix with polygonal stromal cells scattered throughout. These human benign tumours are a valuable research model because of the rarity of cardiac tumours, their clinical presentation and uncertain origin. Here, we assessed whether multipotent cardiac stem/progenitor cells (CSCs) give rise to atrial myxoma tissue. METHODS AND RESULTS: Twenty-three myxomas were collected and analysed for the presence of multipotent CSCs. We detected myxoma cells positive for c-kit (c-kitpos) but very rare Isl-1 positive cells. Most of the c-kitpos cells were blood lineage-committed CD45pos/CD31pos cells. However, c-kitpos/CD45neg/CD31neg cardiac myxoma cells expressed stemness and cardiac progenitor cell transcription factors. Approximately ≤10% of the c-kitpos/CD45neg/CD31neg myxoma cells also expressed calretinin, a characteristic of myxoma stromal cells. In vitro, the c-kitpos/CD45neg/CD31neg myxoma cells secrete chondroitin-6-sulfate and hyaluronic acid, which are the main components of gelatinous myxoma matrix in vivo. In vitro, c-kitpos/CD45neg/CD31neg myxoma cells have stem cell properties being clonogenic, self-renewing, and sphere forming while exhibiting an abortive cardiac differentiation potential. Myxoma-derived CSCs possess a mRNA and microRNA transcriptome overall similar to normal myocardium-derived c-kitpos/CD45neg/CD31negCSCs , yet showing a relatively small and relevant fraction of dysregulated mRNA/miRNAs (miR-126-3p and miR-335-5p, in particular). Importantly, myxoma-derived CSCs but not normal myocardium-derived CSCs, seed human myxoma tumours in xenograft's in immunodeficient NOD/SCID mice. CONCLUSION: Myxoma-derived c-kitpos/CD45neg/CD31neg CSCs fulfill the criteria expected of atrial myxoma-initiating stem cells. The transcriptome of these cells indicates that they belong to or are derived from the same lineage as the atrial multipotent c-kitpos/CD45neg/CD31neg CSCs. Taken together the data presented here suggest that human myxomas could be the first-described CSC-related human heart disease.

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1-2% of total c-kitpos myocardial cells, when stimulated with TGF-ß/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

The adult heart responds to increased workload with physiologic hypertrophy, cardiac stem cell activation, and new myocyte formation.

AIMS: It is a dogma of cardiovascular pathophysiology that the increased cardiac mass in response to increased workload is produced by the hypertrophy of the pre-existing myocytes. The role, if any, of adult-resident endogenous cardiac stem/progenitor cells (eCSCs) and new cardiomyocyte formation in physiological cardiac remodelling remains unexplored. METHODS AND RESULTS: In response to regular, intensity-controlled exercise training, adult rats respond with hypertrophy of the pre-existing myocytes. In addition, a significant number (∼7%) of smaller newly formed BrdU-positive cardiomyocytes are produced by the exercised animals. Capillary density significantly increased in exercised animals, balancing cardiomyogenesis with neo-angiogenesis. c-kit(pos) eCSCs increased their number and activated state in exercising vs. sedentary animals. c-kit(pos) eCSCs in exercised hearts showed an increased expression of transcription factors, indicative of their commitment to either the cardiomyocyte (Nkx2.5(pos)) or capillary (Ets-1(pos)) lineages. These adaptations were dependent on exercise duration and intensity. Insulin-like growth factor-1, transforming growth factor-ß1, neuregulin-1, bone morphogenetic protein-10, and periostin were significantly up-regulated in cardiomyocytes of exercised vs. sedentary animals. These factors differentially stimulated c-kit(pos) eCSC proliferation and commitment in vitro, pointing to a similar role in vivo. CONCLUSION: Intensity-controlled exercise training initiates myocardial remodelling through increased cardiomyocyte growth factor expression leading to cardiomyocyte hypertrophy and to activation and ensuing differentiation of c-kit(pos) eCSCs. This leads to the generation of new myocardial cells. These findings highlight the endogenous regenerative capacity of the adult heart, represented by the eCSCs, and the fact that the physiological cardiac adaptation to exercise stress is a combination of cardiomyocyte hypertrophy and hyperplasia (cardiomyocytes and capillaries).

Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo.

RATIONALE: MicroRNA (miR)-1 and -133 play a crucial role in skeletal and cardiac muscle biology and pathophysiology. However, their expression and regulation in vascular cell physiology and disease is currently unknown. OBJECTIVE: The aim of the present study was to evaluate the role, if any, of miR-1 and miR-133 in vascular smooth muscle cell (VSMC) phenotypic switch in vitro and in vivo. METHODS AND RESULTS: We demonstrate here that miR-133 is robustly expressed in vascular smooth muscle cells (VSMCs) in vitro and in vivo, whereas miR-1 vascular levels are negligible. miR-133 has a potent inhibitory role on VSMC phenotypic switch in vitro and in vivo, whereas miR-1 does not have any relevant effect per se. miR-133 expression is regulated by extracellular signal-regulated kinase 1/2 activation and is inversely correlated with VSMC growth. Indeed, miR-133 decreases when VSMCs are primed to proliferate in vitro and following vascular injury in vivo, whereas it increases when VSMCs are coaxed back to quiescence in vitro and in vivo. miR-133 loss- and gain-of-function experiments show that miR-133 plays a mechanistic role in VSMC growth. Accordingly, adeno-miR-133 reduces but anti-miR-133 exacerbates VSMC proliferation and migration in vitro and in vivo. miR-133 specifically suppresses the transcription factor Sp-1 expression in vitro and in vivo and through Sp-1 repression regulates smooth muscle gene expression. CONCLUSIONS: Our data show that miR-133 is a key regulator of vascular smooth muscle cell phenotypic switch in vitro and in vivo, suggesting its potential therapeutic application for vascular diseases.

Endogenous cardiac stem cell activation by insulin-like growth factor-1/hepatocyte growth factor intracoronary injection fosters survival and regeneration of the infarcted pig heart.

OBJECTIVES: The purpose of this study was to test the ability of insulin-like growth factor (IGF)-1/hepatocyte growth factor (HGF) to activate resident endogenous porcine cardiac stem/progenitor cells (epCSCs) and to promote myocardial repair through a clinically applicable intracoronary injection protocol in a pig model of myocardial infarction (MI) relevant to human disease. BACKGROUND: In rodents, cardiac stem/progenitor cell (CSC) transplantation as well as in situ activation through intramyocardial injection of specific growth factors has been shown to result in myocardial regeneration after acute myocardial infarction (AMI). METHODS: Acute MI was induced in pigs by a 60-min percutaneous transluminal coronary angiography left anterior descending artery occlusion. The IGF-1 and HGF were co-administered through the infarct-related artery in a single dose (ranging from 0.5 to 2 µg HGF and 2 to 8 µg IGF-1) 30 min after coronary reperfusion. Pigs were sacrificed 21 days later for dose-response relationship evaluation by immunohistopathology or 2 months later for cardiac function evaluation by cardiac magnetic resonance imaging. RESULTS: The IGF-1/HGF activated c-kit positive-CD45 negative epCSCs and increased their myogenic differentiation in vitro. The IGF-1/HGF, in a dose-dependent manner, improved cardiomyocyte survival, and reduced fibrosis and cardiomyocyte reactive hypertrophy. It significantly increased c-kit positive-CD45 negative epCSC number and fostered the generation of new myocardium (myocytes and microvasculature) in infarcted and peri-infarct/border regions at 21 and 60 days after AMI. The IGF-1/HGF reduced infarct size and improved left ventricular function at 2 months after AMI. CONCLUSIONS: In an animal model of AMI relevant to the human disease, intracoronary administration of IGF-1/HGF is a practical and effective strategy to reduce pathological cardiac remodeling, induce myocardial regeneration, and improve ventricular function.

Cardiac stem and progenitor cell identification: different markers for the same cell?

For a long time the heart has been considered a terminally differentiated organ without any regenerative potential. The latter has been classically based on the terminally differentiated nature of cardiomyocytes and the absence of a pool of tissue-specific stem cells. This view has been radically changed due to the discovery of resident cardiac stem and progenitor cells in the adult mammalian heart. However, at minimum, 5 apparently different cardiac stem and/or progenitor cell types have been described so far. Thus, we have changed from a view of the heart as a static tissue to an organ with the highest number of tissue-specific stem cell populations. Most likely, the different putative adult cardiac stem and progenitor cells represent different developmental and/or physiological stages of a unique resident adult cardiac stem cell. Notably, it is not yet known the origin of all these cells. A better understanding of the origin, biology and physiology of the myocardial stem and progenitor cells will impact the development of regenerative medicine as an effective therapy for heart disease and failure.

Differential regulation of vascular smooth muscle and endothelial cell proliferation in vitro and in vivo by cAMP/PKA-activated p85alphaPI3K.

cAMP inhibits proliferation in most cell types, triggering different and sometimes opposing molecular pathways. p85alpha (phosphatidylinositol 3-kinase regulatory subunit) is phosphorylated by cAMP/PKA in certain cell lineages, but its effects on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are unknown. In the present study, we evaluated 1) the role of p85alpha in the integration of cAMP/PKA-dependent signaling on the regulation of VSMC and EC growth in vitro; and 2) the effects of PKA-modified p85alpha on neointimal hyperplasia and endothelial healing after balloon injury in vivo. Plasmid constructs carrying wild-type and PKA-modified p85alpha were employed in VSMCs and ECs in vitro and after balloon injury in rat carotid arteries in vivo. cAMP/PKA reduced VSMC proliferation through p85alpha phosphorylation. Transfected PKA-activated p85alpha binds p21ras, reducing ERK1/2 activation and VSMC proliferation in vitro. In contrast, EC proliferation inhibition by cAMP is independent from PKA modification of p85alpha and ERK1/2 inhibition; indeed, PKA-activated p85alpha did not inhibit per se ERK1/2 activation and proliferation in ECs in vitro. Interestingly, cAMP reduced both VSMC and EC apoptotic death through p85alpha phosphorylation. Accordingly, PKA-activated p85alpha triggered Akt activation, reducing both VSMC and EC apoptosis in vitro. Finally, compared with controls, vascular gene transfer of PKA-activated p85alpha significantly reduced neointimal formation after balloon injury in rats, without inhibiting endothelial regeneration of the injured arterial segment. In conclusions, PKA-activated p85alpha integrates cAMP/PKA signaling differently in VSMCs and ECs. By reducing neointimal hyperplasia without inhibiting endothelial regeneration, it exerts a protective effect against restenosis after balloon injury.