Sabrina Valente

Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics.

BACKGROUND AIMSnThe presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue.nnnMETHODSnResident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitro study of the stemness features and multipotency of these multidistrict MSC populations.nnnRESULTSnNotch-1+, Stro-1+, Sca-1+ and Oct-4+ cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothelial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high efflux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis.nnnCONCLUSIONSnOverall, these findings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem Cell Transplantation

Possible cardiac repair by adult stem cell transplantation is currently hampered by poor cell viability and delivery efficiency, uncertain differentiating fate in vivo, the needs of ex vivo cell expansion, and consequent delay in transplantation after the onset of heart attack. By the aid of magnetic resonance imaging, positron emission tomography, and immunohistochemistry, we show that injection of a hyaluronan mixed ester of butyric and retinoic acid (HBR) into infarcted rat hearts afforded substantial cardiovascular repair and recovery of myocardial performance. HBR restored cardiac [18F]fluorodeoxyglucose uptake and increased capillary density and led to the recruitment of endogenous Stro-1-positive stem cells. A terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay demonstrated that HBR-treated hearts exhibited a decrease in the number of apoptotic cardiomyocytes. In isolated rat cardiomyocytes and Stro-1 stem cells, HBR enhanced the transcription of vascular endothelial growth factor, hepatocyte growth factor, kdr, akt, and pim-1. HBR also increased the secretion of vascular endothelial growth factor and hepatocyte growth factor, suggesting that the mixed ester may have recruited both myocardial and Stro-1 cells also. An increase in capillarogenesis was induced in vitro with medium obtained from HBR-exposed cells. In the infarcted myocardium, HBR injection increased histone H4 acetylation significantly. Acetyl-H4 immunoreactivity increased in rat cardiomyocytes and Stro-1 cells exposed to HBR, compared with untreated cells. In conclusion, efficient cardiac regenerative therapy can be afforded by HBR without the need of stem cell transplantation or vector-mediated gene delivery.

Mesenchymal Stem Cells and Islet Cotransplantation in Diabetic Rats: Improved Islet Graft Revascularization and Function by Human Adipose Tissue-Derived Stem Cells Preconditioned With Natural Molecules

Hypoxia plays an important role in limiting the engraftment, survival, and function of intrahepatically transplanted islets. Mesenchymal stem cells (MSCs) were recently used in animal models of islet transplantation not only to reduce allograft rejection but also to promote revascularization. Among different possible origins, adipose tissue represents a novel and good source of MSCs. Moreover, the capability of adipose tissue-derived stem cells (ASCs) to improve islet graft revascularization was recently reported after hybrid transplantation in mice. Within this context, we have previously shown that hyaluronan esters of butyric and retinoic acids can significantly enhance the rescuing potential of human MSCs (hMSCs). Here we evaluated whether ex vivo preconditioning of human ASCs (hASCs) with a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids may result in optimization of graft revascularization after islet/stem cell intrahepatic cotransplantation in syngeneic diabetic rats. We demonstrated that hASCs exposed to the mixture of molecules are able to increase the secretion of vascular endothelial growth factor (VEGF) as well as the transcription of angiogenic genes, including VEGF, KDR (kinase insert domain receptor), and hepatocyte growth factor (HGF). Rats transplanted with islets cocultured with preconditioned hASCs exhibited a better glycemic control than rats transplanted with an equal volume of islets and control hASCs. Cotransplantation with preconditioned hASCs was also associated with enhanced islet revascularization in vivo, as highlighted by graft morphological analysis. The observed increase in islet graft revascularization and function suggests that our method of stem cell preconditioning may represent a novel strategy to remarkably improve the efficacy of islets-hMSCs cotransplantation.

Isolation of stem/progenitor cells from normal lung tissue of adult humans.

Objectives:u2002 This study aimed to isolate and characterize stem/progenitor cells, starting from normal airway epithelia, obtained from human adults.

Mesenchymal stem cells in renal function recovery after acute kidney injury: use of a differentiating agent in a rat model.

Acute kidney injury (AKI) is a major health care condition with limited current treatment options. Within this context, stem cells may provide a clinical approach for AKI. Moreover, a synthetic compound previously developed, hyaluronan monoesters with butyric acid (HB), able to induce metanephric differentiation, formation of capillary-like structures, and secretion of angiogenic cytokines, was tested in vitro. Thereafter, we investigated the effects of human mesenchymal stem cells from fetal membranes (FMhMSCs), both treated and untreated with HB, after induction of ischemic AKI in a rat model. At reperfusion following 45-min clamping of renal pedicles, each rat was randomly assigned to one of four groups: CTR, PBS, MSC, and MSC-HB. Renal function at 1, 3, 5, and 7 days was assessed. Histological samples were analyzed by light and electron microscopy and renal injury was graded. Cytokine analysis on serum samples was performed. FMhMSCs induced an accelerated renal functional recovery, demonstrated by biochemical parameters and confirmed by histology showing that histopathological alterations associated with ischemic injury were less severe in cell-treated kidneys. HB-treated rats showed a minor degree of inflammation, both at cytokine and TEM analyses. Better functional and morphological recovery were not associated to stem cells regenerative processes, but possibly suggest paracrine effects on microenvironment that induce retrieval of renal damaged tissues. These results suggest that FMhMSCs could be useful in the treatment of AKI and the utilization of synthetic compounds could enhance the recovery induction ability of cells.

Poly(butylene/diethylene glycol succinate) multiblock copolyester as a candidate biomaterial for soft tissue engineering: Solid-state properties, degradability, and biocompatibility

A multiblock bioresorbable copolyester, poly(butylene/diethylene glycol succinate), was synthesized by reactive blending, and it was used, together with the corresponding poly(butylene succinate) homopolymer, to form films and to fabricate biomimetic electrospun scaffolds. The poly(butylene/diethylene glycol succinate) scaffold had a more pronounced elastomeric behavior than poly(butylene succinate). It also underwent hydrolytic degradation faster than poly(butylene succinate) since the incorporated diethylene glycol succinate units rendered the copolymer more hydrophilic than poly(butylene succinate). The films degraded faster than electrospun samples due to the autocatalytic effect of carboxylic end-groups. The biodegradable poly(butylene/diethylene glycol succinate) scaffold supported the growth and preserved the cardiac phenotype markers of H9c2 cells, demonstrating its potential utility in soft tissue engineering applications.

Rosuvastatin elicits KDR-dependent vasculogenic response of human placental stem cells through PI3K/AKT pathway.

The growth and plasticity of engrafted human mesenchymal stem cells is regulated by external stimuli. Rosuvastatin (RSV) promotes myocardial neovascularization and limits myocardial remodeling in patients with chronic heart failure (CHF). While these non-lipid benefits may in part depend on the activation of stem cells, experimental evidence that RSV directly elicits vasculogenic differentiation of human mesenchymal stem cells is still lacking. We assessed whether RSV may drive a gene program of vascular commitment and the secretion of trophic mediators with antiapoptotic, angiogenic and antifibrotic activities in human mesenchymal stem cells from full-term placentas (FMhMSCs). With real-time RT-PCR, immunofluorescence, chemiluminescence, Western blot analysis, and in vitro vasculogenesis assays, we show that RSV enhanced expression of vascular endothelial growth factor (VEGF), kinase insert domain receptor (KDR), encoding a major VEGF receptor, hepatocyte growth factor (HGF), and platelet-derived growth factor-BB (PDGF-BB) in a time- and dose-dependent manner. GATA-4 and Nkx-2.5 transcription was not affected. RSV enhanced capillary-like formation in vitro, but capillary-embedded FMhMSCs lacked endothelial marker expression, suggesting a role of pericyte-like elements in tube formation. In HUVEC/FMhMSC cocultures, RSV increases PDGFRβ expression in FMhMSCs, and enhanced capillary density and organizational efficiency, promoting a long-lasting survival of tubular networks. RSV also activated PI3K-Akt pathway; the vasculogenic effects of the statin were abrogated following PI3K inhibition by LY294002. In conclusion, RSV-induced increase in capillary formation was dependent on VEGF and KDR. RSV promotes the activation of paracrine signals for vascular commitment of FMhMSCs through PI3K-Akt pathway. This observation may pave the way to the use of RSV as a pharmacological enhancer of stem cell potential for cardiovascular cell therapy.

The role of 3D microenvironmental organization in MCF-7 epithelial-mesenchymal transition after 7 culture days.

We present a multi-technique study on in vitro epithelial-mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(l-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, β-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, β-catenin and vimentin) and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and β-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-β2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold.

Ultrastructural Assessment of the Differentiation Potential of Human Multipotent Mesenchymal Stromal Cells

Abstract Mesenchymal stromal (stem) cells (MSCs) are defined by plastic adherent growth, multiple phenotype expressions, and tripotential mesodermal capability. The authors report examples where electron microscopy (EM) plays a role in stem cell research. MSCs isolated from human arteries are ultrastructurally heterogeneous and become more homogenous after plastic adhesion. EM shows a moderate complement of organelles, mainly mitochondria, rough endoplasmic reticulum, and glycogen aggregates. Clear vacuoles and vesicles are prominent when cells are recovered from plates using an enzymatic method. Since the mesengenic plasticity is the single most important criterion to define a cell as mesenchymal stromal, the authors induced experimentally adipogenic, leiomyogenic, cardiomyogenic, osteo-chondrogenic differentiations. In no case did EM reveal the achievement of complete differentiation. The authors obtained multivacuolated pre-adipocytes and never univacuolated adipocytes typical of mature white fat; myofibroblast and rhabdomyoblast morphotypes, where contractile filaments were not organized to form functional complexes, i.e., dense bodies and sarcomeres. Chondrogenesis and osteogenesis assays resulted in extracellular matrix changes. Collagen and proteoglycan filament/particle deposition was seen when chondrogenesis was promoted. Hydroxyapatite crystals, psammoma bodies, and plaque-like calcified matrix deposits were found in the osteogenic matrix. EM provides detailed structural information on the degree of differentiation induced in stem cells and demonstrates that the methods so far developed are not able to promote complete cell differentiation. These observations contribute to explain why clinical applications with hMSCs have produced results far lower than initial expectations.

The emerging issue of human resident arterial progenitors: the contribution of organ culture.

Human femoral arteries were cultured up to 56 days. Samples were processed for light, immunohistochemical, and transmission electron microscopy. Arteries became rapidly depopulated; at day 42, an endothelial lining (CD31+, Weibel-Palade bodies) developed on the intima; endothelium was in continuity with mesenchymal stromal cells (CD44+, CD90low, CD105low) placed on adventitia. The media–adventitia area showed heterogeneous cell populations. In long-term organ culture, femoral artery develops a continuous cell coverage that differentiates to endothelium on the intima exclusively. This suggests that distinct topographical factors, such as resident progenitors and/or matrix signals, are able to regulate vascular homeostasis in adult life.