Search

The potential therapeutic effect of extracellular vesicles (EVs) that are derived from human liver stem cells (HLSCs) has been tested in an in vivo model of renal ischemia and reperfusion injury (IRI), that induce the development of chronic kidney disease (CKD). EVs were administered intravenously immediately after the IRI and three days later, then their effect was tested at different time points to evaluate how EV-treatment might interfere with fibrosis development. In IRI-mice that were sacrificed two months after the injury, EV- treatment decreased the development of interstitial fibrosis at the histological and molecular levels. Furthermore, the expression levels of pro-inflammatory genes and of epithelial-mesenchymal transition (EMT) genes were significantly reverted by EV-treatment. In IRI-mice that were sacrificed at early time points (two and three days after the injury), functional and histological analyses showed that EV-treatment induced an amelioration of the acute kidney injury (AKI) that was induced by IRI. Interestingly, at the molecular level, a reduction of pro-fibrotic and EMT-genes in sacrificed IRI-mice was observed at days two and three after the injury. These data indicate that in renal IRI, treatment with HLSC-derived EVs improves AKI and interferes with the development of subsequent CKD by modulating the genes that are involved in fibrosis and EMT.

Human liver stem-cell-derived extracellular vesicles (HLSC-EVs) exhibit therapeutic properties in various pre-clinical models of kidney injury. We previously reported an overall improvement in kidney function following treatment with HLSC-EVs in a model of aristolochic acid nephropathy (AAN). Here, we provide evidence that HLSC-EVs exert anti-fibrotic effects by interfering with β-catenin signalling. A mouse model of AAN and an in vitro pro-fibrotic model were used. The β-catenin mRNA and protein expression, together with the pro-fibrotic markers α-SMA and collagen 1, were evaluated in vivo and in vitro following treatment with HLSC-EVs. Expression and functional analysis of miR29b was performed in vitro following HLSC-EV treatments through loss-of-function experiments. Results showed that expression of β-catenin was amplified both in vivo and in vitro, and β-catenin gene silencing in fibroblasts prevented AA-induced up-regulation of pro-fibrotic genes, revealing that β-catenin is an important factor in fibroblast activation. Treatment with HLSC-EVs caused increased expression of miR29b, which was significantly inhibited in the presence of α-amanitin. The suppression of the miR29b function with a selective inhibitor abolished the anti-fibrotic effects of HLSC-EVs, resulting in the up-regulation of β-catenin and pro-fibrotic α-Sma and collagen type 1 genes. Together, these data suggest a novel HLSC-EV-dependent regulatory mechanism in which β-catenin is down regulated by HLSC-EVs-induced miR29b expression.

Extracellular vesicles (EVs) are membrane vesicles released virtually by all cell types. Several studies have shown that stem cell-derived EVs may mimic both in vitro and in vivo the biological effects of the cells. We recently demonstrated that non-alcoholic steatohepatitis (NASH) is inhibited by treatment with human liver stem cells (HLSCs). The aim of the present study was to evaluate whether EVs released by HLSCs influence the progression of NASH, induced by a diet deprived of methionine and choline, in immunocompromised mice. EV treatment was initiated after 2 weeks of diet with a biweekly administration of three different doses. Bio-distribution evaluated by optical imaging showed a preferential accumulation in normal and, in particular, in fibrotic liver. EV treatment significantly improved liver function and reduced signs of liver fibrosis and inflammation at both morphological and molecular levels. In particular, we observed that, out of 29 fibrosis-associated genes upregulated in NASH liver, 28 were significantly downregulated by EV treatment. In conclusion, HLSC-derived EVs display anti-fibrotic and anti-inflammatory effects in a model of chronic liver disease, leading to an improvement of liver function., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular vesicles released into urine (uEVs) can represent interesting biomarkers of renal cell damage. CD133, a stem/progenitor cell marker expressed by renal progenitor cells, is highly expressed in uEVs of healthy individuals. In the present study, we evaluated the level of CD133 in the uEVs of patients with acute and chronic glomerular damage by cytofluorimetric analysis. The level of CD133 + uEVs was significantly decreased in pediatric patients with acute glomerulonephritis during the acute phase of renal damage, while it was restored after the subsequent recovery. A similar decrease was also observed in patients with chronic glomerulonephritis. Moreover, CD133 + uEVs significantly declined in patients with type 2 diabetes, used as validation group, with the lowest levels in patients with albuminuria with diabetic nephropathy. Indeed, receiver-operating characteristic curve analysis indicates the ability of CD133 + uEV values to discriminate the health condition from that of glomerular disease. In parallel, a significant decrease of CD133 in renal progenitor cells and in their derived EVs was observed in vitro after cell treatment with a combination of glucose and albumin overload, mimicking the diabetic condition. These data indicate that the level of CD133 + uEVs may represent an easily accessible marker of renal normal physiology and could provide information on the "reservoir" of regenerating cells within tubules.

Crigler Najjar Syndrome type I (CNSI) is a rare recessive disorder caused by mutations in the Ugt1a1 gene. There is no permanent cure except for liver transplantation, and current therapies present several shortcomings. Since stem cell-based therapy offers a promising alternative for the treatment of this disorder, we evaluated the therapeutic potential of human liver stem cells (HLSC) in immune-compromised NOD SCID Gamma (NSG)/Ugt1 -/- mice, which closely mimic the pathological manifestations in CNSI patients. To assess whether HLSC expressed UGT1A1, decellularised mouse liver scaffolds were repopulated with these cells. After 15 days' culture ex vivo, HLSC differentiated into hepatocyte-like cells showing UGT1A1 expression and activity. For the in vivo human cell engraftment and recovery experiments, DiI-labelled HLSC were injected into the liver of 5 days old NSG/Ugt1 -/- pups which were analysed at postnatal Day 21. HLSC expressed UGT1A1 in vivo, induced a strong decrease in serum unconjugated bilirubin, thus significantly improving phenotype and survival compared to untreated controls. A striking recovery from brain damage was also observed in HLSC-injected mutant mice versus controls. Our proof-of-concept study shows that HLSC express UGT1A1 in vivo and improve the phenotype and survival of NSG/Ugt1 -/- mice, and show promises for the treatment of CNSI.

Objectives: Critical hindlimb ischemia is a severe consequence of peripheral artery disease. Surgical treatment does not prevent skeletal muscle impairment or improve long-term patient outcomes. The present study investigates the protective/regenerative potential and the mechanism of action of adipose stem cell-derived extracellular vesicles (ASC-EVs) in a mouse model of hindlimb ischemia. Approach and Results: We demonstrated that ASC-EVs exert a protective effect on muscle damage by acting both on tissue microvessels and muscle cells. The genes involved in muscle regeneration were up-regulated in the ischemic muscles of ASC-EV-treated animals. MyoD expression has also been confirmed in satellite cells. This was followed by a reduction in muscle function impairment in vivo. ASC-EVs drive myoblast proliferation and differentiation in the in vitro ischemia/reoxygenation model. Moreover, ASC-EVs have shown an anti-apoptotic effect both in vitro and in vivo. Transcriptomic analyses have revealed that ASC-EVs carry a variety of pro-angiogenic mRNAs, while proteomic analyses have demonstrated an enrichment of NRG1 (neuregulin 1). A NRG1 blocking antibody used in vivo demonstrated that NRG1 is relevant to ASC-EV-induced muscle protection, vascular growth, and recruitment of inflammatory cells. Finally, bioinformatic analyses on 18 molecules that were commonly detected in ASC-EVs, including mRNAs and proteins, confirmed the enrichment of pathways involved in vascular growth and muscle regeneration/protection., Conclusions: This study demonstrates that ASC-EVs display pro-angiogenic and skeletal muscle protective properties that are associated with their NRG1/mRNA cargo. We, therefore, propose that ASC-EVs are a useful tool for therapeutic angiogenesis and muscle protection.

Insulin-dependent diabetes mellitus or type 1 diabetes mellitus (T1DM) is an auto-immune condition characterized by the loss of pancreatic β-cells. The curative approach for highly selected patients is the pancreas or the pancreatic islet transplantation. Nevertheless, these options are limited by a growing shortage of donor organs and by the requirement of immunosuppression.Xenotransplantation of porcine islets has been extensively investigated. Nevertheless, the strong xenoimmunity and the risk of transmission of porcine endogenous retroviruses, have limited their application in clinic. Generation of β-like cells from stem cells is one of the most promising strategies in regenerative medicine. Embryonic, and more recently, adult stem cells are currently the most promising cell sources exploited to generate functional β-cells in vitro. A number of studies demonstrated that stem cells could generate functional pancreatic organoids (POs), able to restore normoglycemia when implanted in different preclinical diabetic models. Nevertheless, a gradual loss of function and cell dead are commonly detected when POs are transplanted in immunocompetent animals. So far, the main issue to be solved is the post-transplanted islet loss, due to the host immune attack. To avoid this hurdle, nanotechnology has provided a number of polymers currently under investigation for islet micro and macro-encapsulation. These new approaches, besides conferring PO immune protection, are able to supply oxygen and nutrients and to preserve PO morphology and long-term viability.Herein, we summarize the current knowledge on bioengineered POs and the stem cell differentiation platforms. We also discuss the in vitro strategies used to generate functional POs, and the protocols currently used to confer immune-protection against the host immune attack (micro- and macro-encapsulation). In addition, the most relevant ongoing clinical trials, and the most relevant hurdles met to move towards clinical application are revised.

The extracellular vesicles (EVs) are membrane vesicles carrying proteins, nucleic acids, and bioactive lipids of the cell of origin. These vesicles released within the extracellular space and entering into the circulation may transfer their cargo to neighboring or distant cells and induce phenotypical and functional changes that may be relevant in several physiopathological conditions. In an attempt to define the biological properties of EVs, several investigations have focused on their cargo and on the effects elicited in recipient cells. EVs have been involved in modulation of tumor microenvironment and behavior, as well as in the immune and inflammatory response. In the present review, we address the paracrine action of EVs released by stem cells and their potential involvement in the activation of regenerative programs in injured cells.

A potential therapeutic strategy for diabetes is the transplantation of induced-insulin secreting cells. Based on the common embryonic origin of liver and pancreas, we studied the potential of adult human liver stem-like cells (HLSC) to generate in vitro insulin-producing 3D spheroid structures (HLSC-ILS). HLSC-ILS were generated by a one-step protocol based on charge dependent aggregation of HLSC induced by protamine. 3D aggregation promoted the spontaneous differentiation into cells expressing insulin and several key markers of pancreatic β cells. HLSC-ILS showed endocrine granules similar to those seen in human β cells. In static and dynamic in vitro conditions, such structures produced C-peptide after stimulation with high glucose. HLSC-ILS significantly reduced hyperglycemia and restored a normo-glycemic profile when implanted in streptozotocin-diabetic SCID mice. Diabetic mice expressed human C-peptide and very low or undetectable levels of murine C-peptide. Hyperglycemia and a diabetic profile were restored after HLSC-ISL explant. The gene expression profile of in vitro generated HLSC-ILS showed a differentiation from HLSC profile and an endocrine commitment with the enhanced expression of several markers of β cell differentiation. The comparative analysis of gene expression profiles after 2 and 4 weeks of in vivo implantation showed a further β-cell differentiation, with a genetic profile still immature but closer to that of human islets. In conclusion, protamine-induced spheroid aggregation of HLSC triggers a spontaneous differentiation to an endocrine phenotype. Although the in vitro differentiated HLSC-ILS were immature, they responded to high glucose with insulin secretion and in vivo reversed hyperglycemia in diabetic SCID mice.

Human liver stem-like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti-tumor activity. In our study, we investigated whether HLSC-derived EVs (HLSC-EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC-EVs). The results obtained indicated that HLSC-EVs, but not MSC-EVs, inhibited the angiogenic properties of tumor-derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC-EVs led to the down-regulation of pro-angiogenic genes. Since HLSC-EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC-EV specific miRNAs. We observed that four miRNAs, namely miR-15a, miR-181b, miR-320c and miR-874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC-EVs significantly enhanced expression of miR-15a, miR-181b, miR-320c and miR-874 associated with the down-regulation of FGF1 and PLAU. In summary, HLSC-EVs possess an anti-tumorigenic effect, based on their ability to inhibit tumor angiogenesis., (© 2018 UICC.)

Stem cells act in a paracrine manner through the secretion of biologically active cargo that acts on cells locally and systemically. These active molecules include not only soluble factors but also extracellular vesicles (EVs) that have recently emerged as a mechanism of cell-to-cell communication. EVs act as vehicles that transfer molecules between originator and recipient cells, thereby modifying the phenotype and function of the latter. As EVs released from stem cells may successfully activate regenerative processes in injured cells, their application as a form of therapy can be envisaged. EVs exert these proregenerative effects through the modulation of relevant cellular processes including proliferation, angiogenesis, oxidative stress, inflammation, and immunotolerance, among others. In this chapter, we review the preclinical studies that report the effect of stem cell-derived EVs in various pathological models of human disease.

Background: The renal assist device (RAD) is a blood purification system containing viable renal tubular epithelial cells (TECs) that has been proposed for the treatment of acute kidney injury (AKI) and multiple organ failure. Perfluorocarbons (PFCs) are oxygen carriers used for organ preservation in transplantation. The aim of this study was to investigate the effect of PFCs on hypoxia- and sepsis-induced TEC injury and on renal CD133+ progenitor differentiation in a microenvironment similar to the RAD., Methods: TECs were seeded in a polysulphone hollow fibre under hypoxia or cultured with plasma from 10 patients with sepsis-associated AKI in the presence or absence of PFCs and were tested for cytotoxicity (XTT assay), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling assay, caspases, enzyme-linked immunosorbent assay, Fas/Fas Ligand pathway activation), mitochondrial activity, cell polarity [transepithelial electrical resistance (TEER)] and adenosine triphosphate production. The effect of PFCs on proliferation and differentiation of human CD133+ progenitors was also studied., Results: In the presence of PFCs, TECs seeded into the polysulphone hollow fibre showed increased viability and expression of insulin-like growth factor 1, hepatocyte growth factor and macrophage-stimulating protein. Plasma from septic patients induced TEC apoptosis, disruption of oxidative metabolism, alteration of cell polarity and albumin uptake, down-regulation of the tight junction protein ZO-1 and the endocytic receptor megalin on the TEC surface. These detrimental effects were significantly reduced by PFCs. Moreover, PFCs induced CD133+ renal progenitor cell proliferation and differentiation towards an epithelial/tubular-like phenotype., Conclusions: PFCs improved the viability and metabolic function of TECs seeded within a polysulphone hollow fibre and subjected to plasma from septic AKI patients. Additionally, PFCs promoted differentiation towards a tubular/epithelial phenotype of CD133+ renal progenitor cells.

Background: Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia., Methods: HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells., Results: Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme and mRNA. In fact, EVs from ASS1-knockdown HLSCs contained low amounts of ASS1 mRNA and protein, and were unable to restore urea production in hepatocytes differentiated from ASS1-HLSCs., Conclusions: Collectively, these results suggest that EVs derived from normal HLSCs may compensate the loss of ASS1 enzyme activity in hepatocytes differentiated from ASS1-HLSCs.

Persistent alterations of the renal tissue due to maladaptive repair characterize the outcome of acute kidney injury (AKI), despite a clinical recovery. Acute damage may also limit the renal production of erythropoietin, with impairment of the hemopoietic response to ischemia and possible lack of its reno-protective action. We aimed to evaluate the effect of a cell therapy using human CD133 + renal progenitor cells on maladaptive repair and fibrosis following AKI in a model of glycerol-induced rhabdomyolysis. In parallel, we evaluated the effect of CD133 + cells on erythropoietin production. Administration of CD133 + cells promoted the restoration of the renal tissue, limiting the presence of markers of injury and pro-inflammatory molecules. In addition, it promoted angiogenesis and protected against fibrosis up to day 60. No effect of dermal fibroblasts was observed. Treatment with CD133 + cells, but not with PBS or fibroblasts, limited anemia and increased erythropoietin levels both in renal tissue and in circulation. Finally, CD133 + cells contributed to the local production of erythropoietin, as observed by detection of circulating human erythropoietin. CD133 + cells appear therefore an effective source for cell repair, able to restore renal functions, including erythropoietin release, and to limit long term maldifferentiation and fibrosis.

Extracellular vesicles (EVs) that are derived from stem cells are proving to be promising therapeutic options. We herein investigate the therapeutic potential of EVs that have been derived from different stem cell sources, bone-marrow (MSC) and human liver (HLSC), on mesangial cells (MCs) exposed to hyperglycaemia. By expressing a dominant negative STAT5 construct (ΔNSTAT5) in HG-cultured MCs, we have demonstrated that miR-21 expression is under the control of STAT5, which translates into Transforming Growth Factor beta (TGFβ) expression and collagen production. A number of approaches have been used to show that both MSC- and HLSC-derived EVs protect MCs from HG-induced damage via the transfer of miR-222. This resulted in STAT5 down-regulation and a decrease in miR-21 content, TGFβ expression and matrix protein synthesis within MCs. Moreover, we demonstrate that changes in the balance between miR-21 and miR-100 in the recipient cell, which are caused by the transfer of EV cargo, further contribute to providing beneficial effects. Interestingly, these effects were only detected in HG-cultured cells. Finally, it was found that HG reduced the expression of the nuclear encoded mitochondrial electron transport chain (ETC) components, CoxIV. It is worth noting that EV administration can rescue CoxIV expression in HG-cultured MCs. These results thus demonstrate that both MSC- and HLSC-derived EVs transfer the machinery needed to preserve MCs from HG-mediated damage. This occurs via the horizontal transfer of functional miR-222 which directly interferes with damaging cues. Moreover, our data indicate that the release of EV cargo into recipient cells provides additional therapeutic advantages against harmful mitochondrial signals., Competing Interests: The authors have declared that no competing interests exist.

Evidence indicates that Alix, an accessory protein of the endosomal sorting complex required for transport (ESCRT), is involved in the biogenesis of extracellular vesicles (EVs). EVs contain selected patterns of microRNAs (miRNAs or miRs); however, little is known about the mechanisms of miRNA enrichment in EVs. The aim of the present study was to evaluate whether Alix is involved in the packaging of miRNAs within EVs released by human liver stem‑like cells (HLSCs). EVs released from HLSCs were enriched with miRNAs and expressed Alix and several RNA-binding proteins, including Argonaute 2 (Ago2), a member of the Argonaute family known to be involved in the transport and the processing of miRNAs. Co-immunoprecipitation experiments revealed an association between Alix and Ago2. The results from RT-qPCR indicated that in the Alix/Ago2 immunoprecipitates, miRNAs were detectable. EVs were instrumental in transferring selected miRNAs from HLSCs to human endothelial cells absent in the latter cells. Alix knockdown did not influence the number of EVs released by HLSCs, but it significantly decreased miRNA expression levels in the EVs and consequently their transfer to the endothelium. Our findings indicate that Alix binds to Ago2 and miRNAs, suggesting that it plays a key role in miRNA enrichment during EV biogenesis. These results may represent a novel function of Alix, demonstrating its involvement in the EV-mediated transfer of miRNAs.

2013 saw the publication of numerous studies that identified resident renal stem or progenitor cells, induced pluripotent stem cells and strategies based on stem cell paracrine action, which all might be suitable for kidney regeneration after injury.

The paracrine theory has recently changed the view of the biological action of stem cells and of the subsequent potential application of stem cells in regenerative medicine. Indeed, most of the beneficial effects of stem-cell-based therapy have been attributed to soluble factors released from stem cells. In this context, MVs (microvesicles) released as exosomes from the endosomal compartment, or as shedding vesicles from the cell surface, may play a relevant role in the intercellular communication between stem and injured cells. By transferring proteins, bioactive lipids, mRNA and microRNA, MVs act as vehicles of information that may lead to alteration of the phenotype of recipient cells. The exchange of information between stem cells and tissue-injured cells is reciprocal. The MV-mediated transfer of tissue-specific information from the injured cells to stem cells may reprogramme the latter to gain phenotypic and functional characteristics of the cell of origin. On the other hand, MVs released from stem cells may confer a stem-cell-like phenotype to injured cells, with the consequent activation of self-regenerative programmes. In fact, MVs released from stem cells retain several biological activities that are able to reproduce the beneficial effects of stem cells in a variety of experimental models.

There has been increasing interest in the application of mesenchymal stem cells (MSCs) in regenerative medicine in recent years. In this context, the beneficial effects of MSCs have been ascribed mainly to a paracrine action rather than to direct replacement of the injured tissue. Indeed, MSCs produce a great variety of trophic and immunomodulatory factors. In this chapter, we provide an overview of growth factors and chemokines involved in stimulation of cell proliferation, inhibition of apoptosis, enhancement of angiogenesis, and suppression of inflammatory and immune response. In addition, we discuss the emerging role of the extracellular vesicles released from MSCs as possible paracrine mediators.

Endothelial progenitor cells are known to reverse acute kidney injury by paracrine mechanisms. We previously found that microvesicles released from these progenitor cells activate an angiogenic program in endothelial cells by horizontal mRNA transfer. Here, we tested whether these microvesicles prevent acute kidney injury in a rat model of ischemia-reperfusion injury. The RNA content of microvesicles was enriched in microRNAs (miRNAs) that modulate proliferation, angiogenesis, and apoptosis. After intravenous injection following ischemia-reperfusion, the microvesicles were localized within peritubular capillaries and tubular cells. This conferred functional and morphologic protection from acute kidney injury by enhanced tubular cell proliferation, reduced apoptosis, and leukocyte infiltration. Microvesicles also protected against progression of chronic kidney damage by inhibiting capillary rarefaction, glomerulosclerosis, and tubulointerstitial fibrosis. The renoprotective effect of microvesicles was lost after treatment with RNase, nonspecific miRNA depletion of microvesicles by Dicer knock-down in the progenitor cells, or depletion of pro-angiogenic miR-126 and miR-296 by transfection with specific miR-antagomirs. Thus, microvesicles derived from endothelial progenitor cells protect the kidney from ischemic acute injury by delivering their RNA content, the miRNA cargo of which contributes to reprogramming hypoxic resident renal cells to a regenerative program.

The efficacy of islet transplantation is limited by poor graft vascularization. We herein demonstrated that microvesicles (MVs) released from endothelial progenitor cells (EPCs) enhanced human islet vascularization. After incorporation into islet endothelium and β-cells, EPC-derived MVs favored insulin secretion, survival, and revascularization of islets transplanted in SCID mice. MVs induced in vitro islet endothelial cell proliferation, migration, resistance to apoptosis, and organization in vessel-like structures. Moreover, MVs partially overcame the antiangiogenic effect of rapamycin and inhibited endothelial-leukocyte interaction via L-selectin and CD40. MVs were previously shown to contain defined patterns of mRNAs. Here we demonstrated that MVs carried the proangiogenic miR-126 and miR-296 microRNAs (miRNAs). MVs pretreated with RNase or derived from Dicer knocked-down EPCs showed a reduced angiogenic effect. In addition, MVs overcame the antiangiogenic effect of the specific antagomiRs of miR-126 and miR-296, suggesting a relevant contribution of miRNAs delivered by MVs to islet endothelium. Microarray analysis of MV-stimulated islet endothelium indicated the upregulation of mRNAs coding for factors involved in endothelial proliferation, differentiation, and angiogenesis. In addition, MVs induced the activation of the PI3K-Akt and eNOS signaling pathways in islet endothelium. These results suggest that MVs activate an angiogenic program in islet endothelium that may sustain revascularization and β-cell function.

Paracrine mediators released from endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis following ischemia. Recently, we demonstrated that microvesicles (MVs) derived from EPCs are able to activate an angiogenic program in quiescent endothelial cells by a horizontal transfer of RNA. In this study we aim to investigate whether EPC-derived MVs are able to induce neoangiogenesis and to enhance recovery in a murine model of hindlimb ischemia. Hindlimb ischemia was induced in severe combined immunodeficient (SCID) mice by ligation and resection of the left femoral artery and mice were treated with EPC-derived MVs (MVs), RNase-inactivated MVs (RnaseMVs), fibroblast-derived MVs or vehicle alone as control (CTL). Since MVs contained the angiogenic miR-126 and miR-296, we evaluated whether microRNAs may account for the angiogenic activities by treating mice with MVs obtained from DICER-knock-down EPC (DICER-MVs). The limb perfusion evaluated by laserdoppler analysis demonstrated that MVs significantly enhanced perfusion in respect to CTL (0.50±0.08 vs 0.39±0.03, p<0.05). After 7 days, immunohistochemical analyses on the gastrocnemius muscle of the ischemic hindlimb showed that MVs but not fibroblast-MVs significantly increased the capillary density in respect to CTL (MVs vs CTL: 24.7±10.3 vs 13.5±6, p<0.0001) and (fibroblast-MVs vs CTL: 10.2±3.4 vs 13.5±6, ns); RNaseMVs and DICER-MVs significantly reduced the effect of MVs (RNaseMVs vs CTL: 15.7±4.1 vs 13.5±6, ns) (MVs vs DICER-MVs 24.7±10.3 vs 18.1±5.8, p <0.05), suggesting a role of RNAs shuttled by MVs. Morphometric analysis confirmed that MVs enhanced limb perfusion and reduced injury. The results of the present study indicate that treatment with EPC-derived MVs improves neovascularization and favors regeneration in severe hindlimb ischemia induced in SCID mice. This suggests a possible use of EPCs-derived MVs for treatment of peripheral arterial disease.

Low-oxygen tension is an important component of the stem cell microenvironment. In rodents, renal resident stem cells have been described in the papilla, a relatively hypoxic region of the kidney. In the present study, we found that CD133(+) cells, previously described as renal progenitors in the human cortex, were enriched in the renal inner medulla and localized within the Henle's loop and thin limb segments. Once isolated, the CD133(+) cell population expressed renal embryonic and stem-related transcription factors and was able to differentiate into mature renal epithelial cells. When injected subcutaneously in immunodeficient mice within Matrigel, CD133(+) cells generated canalized structures positive for renal specific markers of different nephron segments. Oct4A levels and differentiation potential of papillary CD133(+) cells were higher than those of CD133(+) cells from cortical tubuli. Hypoxia was able to promote the undifferentiated phenotype of CD133(+) progenitors from papilla. Hypoxia stimulated clonogenicity, proliferation, vascular endothelial growth factor synthesis, and expression of CD133 that were in turn reduced by epithelial differentiation with parallel HIF-1α downregulation. In addition, hypoxia downregulated microRNA-145 and promoted the synthesis of Oct4A. Epithelial differentiation increased microRNA-145 and reduced Oct4 level, suggesting a balance between Oct4 and microRNA-145. MicroRNA-145 overexpression in CD133(+) cells induced downrelation of Oct4A at the protein level, inhibited cell proliferation, and stimulated terminal differentiation. This study underlines the role of the hypoxic microenvironment in controlling the proliferation and maintaining a progenitor phenotype and stem/progenitor properties of CD133(+) cells of the nephron. This mechanism may be at the basis of the maintenance of a CD133(+) population in the papillary region and may be involved in renal regeneration after injury.

Angiogenesis inhibiting agents are currently integral component of anticancer therapy. However, tumors, initially responsive to anti-angiogenic drugs or vascular targeting agents, can acquire resistance. The limited clinical efficacy might result from the heterogeneous nature of tumors or alternatively from the unique phenotype of tumor vascular cells, widely diverse from so-called 'normal' endothelium. Hence, defining the molecular mechanisms driving this diversity might provide a rational basis to design combinatory therapies that should be more effective in avoiding resistance. Herein, we demonstrated that tumor-derived endothelial cells (TECs) isolated from breast and kidney carcinomas retained an endothelial phenotype, but outspread independently of growth factors. Applying small interfering RNA approach, we demonstrated that interleukin (IL)-3, but not vascular endothelial growth factor, released by TECs, supports their autocrine growth and promotes in vivo vessel formation and tumor angiogenesis. Meanwhile, we found that the expression of the membrane-bound kit ligand (mbKitL) depends on IL-3, and it is crucial for adhesion of endothelial progenitor cells (EPCs) and inflammatory cells to TECs. These events required Akt activation. Finally, the finding that depletion of the mbKitL prevented EPC and inflammatory cell trafficking into vascular microenvironment, indicates that, as in bone marrow, the mbKitL can act as a membrane/adhesion molecule for c-Kit-expressing cells. These data provide evidences that an IL-3 autocrine loop can drive a tumor endothelial switch and that targeting IL-3 might confer a significant therapeutic advantage to hamper tumor angiogenesis.

In autosomal dominant polycystic kidney disease, cysts arise focally and disrupt normal renal tissue leading to renal failure. In the present study, we show that cyst-lining cells express the stem cell marker CD133. CD133+ progenitor cells isolated from polycystic kidney, carrying mutations of PKD genes, showed a dedifferentiated phenotype similar to CD133+ progenitor cells from normal kidney. However, these cells were more proliferative and presented a defective epithelial differentiation phenotype with respect to normal renal CD133+ cells as they were not able to express all tubular epithelial cell markers when cultured in epithelial differentiation medium. Polycystic CD133+ cells, in contrast to normal renal CD133+ cells, formed cysts in vitro in a three-dimensional culture system and in vivo when injected subcutaneously within Matrigel in SCID mice. Rapamycin treatment reduced in vitro proliferation of polycystic CD133+ cells and decreased cystogenesis both in vitro and in vivo. The in vitro epithelial differentiation was only partially improved by rapamycin. These results indicate that polycystic CD133+ cells retain a dedifferentiated phenotype and the ability to generate cysts., (Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Stem cells isolated from human amniotic fluid are gaining attention with regard to their therapeutic potential. In this work, we investigated whether these cells contribute to tubular regeneration after experimental acute kidney injury. Cells expressing stem cell markers with multidifferentiative potential were isolated from human amniotic fluid. The regenerative potential of human amniotic fluid stem cells was compared with that of bone marrow-derived human mesenchymal stem cells. We found that the intravenous injection of 3.5 × 10(5) human amniotic fluid stem cells into nonimmune-competent mice with glycerol-induced acute kidney injury was followed by rapid normalization of renal function compared with injection of mesenchymal stem cells. Both stem cell types showed enhanced tubular cell proliferation and reduced apoptosis. Mesenchymal stem cells were more efficient in inducing proliferation than amniotic fluid-derived stem cells, which, in contrast, were more antiapoptotic. Both cell types were found to accumulate within the peritubular capillaries and the interstitium, but amniotic fluid stem cells were more persistent than mesenchymal stem cells. In vitro experiments demonstrated that the two cell types produced different cytokines and growth factors, suggesting that a combination of different mediators is involved in their biological actions. These results suggest that the amniotic fluid-derived stem cells may improve renal regeneration in acute kidney injury, but they are not more effective than mesenchymal stem cells.

Background: Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs)., Methodology/principal Findings: MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation., Conclusions: This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.

Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MVs) derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MVs in the hepatocytes by an alpha(4)-integrin-dependent mechanism. However, MVs pre-treated with RNase, even if internalized, were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA-dependent effect. Microarray analysis and quantitative RT-PCR demonstrated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MVs accelerated the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre-treatment of MVs. Using human AGO2, as a reporter gene present in MVs, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MVs. These data suggested a translation of the MV shuttled mRNA into hepatocytes of treated rats. In conclusion, these results suggest that MVs derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets.

The use of bioartificial livers (BALs) for the expansion of human adult liver stem cells and the production of growth factors could be a potential strategy for cell-based extracorporeal liver support. The present study aimed to assessing the differentiation of human adult liver stem cells in a rotary BAL. Liver stem cells were seeded into a polysulphone membrane filter at a density of 3 x 10(8) cells, and the filter was connected to a rotary bioreactor perfusion system (37 degrees C, 50 mL/min, 48 h). Viability, cell differentiation, and metabolic performances were evaluated at 24 and 48 h. Hepatocyte growth factor production from human adult liver stem cells, mature hepatocytes, and mesenchymal stem cells in adhesion and in the rotary BAL conditions was compared. Liver stem cells cultured in the rotary BAL produced the highest amounts of albumin (p = 0.002) and ammonia-induced urea (p = 0.0001), and had an increased cytochrome P450 expression in respect to liver stem cells in adhesion. Remarkably, liver stem cells in the rotary BAL produced very high amounts of hepatocyte growth factor (p = 0.005) in respect to hepatocytes and mesenchymal stem cells. Moreover, the cells lost their stem cell markers and acquired several markers of mature hepatocytes. In conclusion, the rotary BAL favored liver stem cell differentiation into mature hepatocyte-like cells.

Breast tumour stem cells have been reported to differentiate in the epithelial lineage but a cross-lineage potential has not been investigated. We aimed to evaluate whether breast tumour stem cells were able to differentiate also into the endothelial lineage. We isolated and cloned a population of breast tumour stem cells, cultured as mammospheres that expressed the stem markers nestin and Oct-4 and not epithelial and endothelial differentiation markers, and formed serially transplantable tumours in SCID mice. When cultured in the presence of serum, mammosphere-derived clones differentiated in the epithelial lineage. When cultured in the presence of VEGF, the same clones were also able to differentiate in the endothelial lineage acquiring endothelial markers and properties, such as the ability to organize in Matrigel into capillary-like structures. In the transplanted tumours, originated from mammospheres, we demonstrate that some of the intratumour vessels were of human origin, suggesting an in vivo endothelial differentiation of mammosphere-derived cells. Finally, endothelial cell clones originated from mammospheres were able, when implanted in Matrigel in SCID mice, to form after 7 days a human vessel network and, after 3-4 weeks, an epithelial tumour suggesting that in the endothelial-differentiated cells a tumourigenic stem cell population is maintained. In conclusion, the results of the present study demonstrate that stem cells of breast cancer have the ability to differentiate not only in epithelial but also in endothelial lineage, further supporting the hypothesis that the tumour-initiating population possesses stem cell characteristics relevant for tumour growth and vascularization.

In the adult kidney, different populations of progenitor cells (or stem cells) have been identified. These cells may represent a remnant of embryonic stem cells in the adult tissue, or populations of bone-marrow-derived stem cells homed within the kidney and modified by the local microenvironment. This modification may be the expression of a partial commitment or of different degrees of maturation. Resident stem cells may account for the growth of the organ during development, for the physiological cell turnover, and for the repair of kidney damage. In addition, stem cells derived from the bone marrow and migrated through the circulation to the site of the damage may contribute to tissue repair. Preliminary studies suggest that this regenerative potential of stem cells could be exploited for therapeutic purposes by administration of ex vivo expanded stem cell populations or by development of strategies aimed to expand and differentiate local stem cells.

The potential role of stem cells in the repair of glomerular and tubular injury is under intensive investigation. Several studies have addressed the role of endogenous bone-marrow (BM)-derived stem cells (SC) in the repair of renal injury. Some reports indicate that BM-derived SC are capable of engraftment into damaged nephrons, although the lineage of SC recruited has not been established. However, this issue remains highly controversial, and several studies point to a paracrine/endocrine action of endogenous SC rather than a direct repopulation of the injured nephron. Administration of exogenous mesenchymal SC in experimental acute kidney injury suggests that an SC-based therapy may improve functional and structural recovery of both glomerular and tubular compartments. A contribution of renal resident SC has also been postulated in the recovery after damage. However, several points still need to be clarified regarding the signals regulating homing of SC to injured tissue, the secreted factors underlying the paracrine/endocrine mechanisms, and in particular the long-term in vivo behavior of the administered SC. This review summarizes the current literature on the physiological role of endogenous SC in renal regeneration and on the potential therapeutic strategies based on the administration of exogenous SC., (Copyright 2008 S. Karger AG, Basel.)

Membrane-derived microvesicles (MVs) are released from the cell surface and are implicated in cell-to-cell communication. We evaluated whether MVs derived from endothelial progenitor cells (EPCs) are able to trigger angiogenesis. We found that EPC-derived MVs were incorporated in endothelial cells by interaction with alpha4 and beta1 integrins expressed on the MV surface. In vitro, MVs promoted endothelial cell survival, proliferation, and organization in capillary-like structures. In vivo, in severe combined immunodeficient (SCID) mice, MV-stimulated human endothelial cells organized in patent vessels. When incubated with RNase, despite their internalization into endothelial cells, MVs failed to induce in vitro and in vivo angiogenic effects. mRNA transfer was shown by transduction of GFP protein in endothelial cells by MVs containing GFP-mRNA and the biologic relevance by the angiogenic effect of MV-mRNA extract delivered by lipofectamine. Microarray ana-lysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) of MV-mRNA extract indicated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated with the PI3K/AKT signaling pathway. Protein expression and functional studies showed that PI3K and eNOS play a critical role in the angiogenic effect of MVs. These results suggest that EPCs may activate angiogenesis in endothelial cells by releasing MVs able to trigger an angiogenic program.

We investigated the role of c-Kit and the membrane-bound ligand (mbKitL) in endothelial progenitor cell (EPC) recruitment by microvascular endothelial cells (ECs). We demonstrated that inflammatory activation induced the expression of the mbKitL on ECs both in vitro and in vivo, and that recruitment of EPCs depended on c-Kit/mbKitL interaction. Depletion of endogenous c-Kit or inhibition of c-Kit enzymatic activity by imatinib mesylate prevented adhesion of EPCs to activated ECs both in vitro and in vivo, indicating that a functional c-Kit on EPCs is essential. We also demonstrate that Akt was the downstream molecule regulating cell adhesion. A potential role of the c-Kit/mbKitL interaction in pathological settings is sustained by the expression of the mbKitL on ECs lining intraplaque neovessels. Thus, our results provide new insights into the mechanisms underlying EPC recruitment and the bases for novel strategies to hinder pathological angiogenesis.

In the present study, we tested the hypothesis that resident progenitor cells may contribute to tumor vascularization and growth. CD133+ cells were isolated from 30 human renal carcinomas and characterized as renal resident progenitor cells on the basis of the expression of renal embryonic and mesenchymal stem cell markers. CD133+ progenitors differentiated into endothelial and epithelial cells as the normal CD133+ counterpart present in renal tissue. In the presence of tumor-derived growth factors, these cells were committed to differentiate into endothelial cells able to form vessels in vivo in SCID mice. Undifferentiated CD133+ progenitors were unable to form tumors when transplanted alone in SCID mice. When co-transplanted with renal carcinoma cells, CD133+ progenitors significantly enhanced tumor development and growth. This effect was not attributable to the tumorigenic nature of CD133+ progenitor cells because the same results were obtained with CD133+ cells from normal kidney. CD133+ progenitors contributed to tumor vascularization as the majority of neoformed vessels present within the transplanted tumors were of human origin and derived from the co-transplanted CD133+ progenitors. In conclusion, these results indicate the presence of a renal progenitor cell population in renal carcinomas that may differentiate in endothelial cells and favor vascularization and tumor growth.

We describe here isolation and characterization of CD133+ cells derived from normal adult human kidney. These cells lacked the expression of hematopoietic markers and expressed PAX-2, an embryonic renal marker, suggesting their renal origin. Renal tissue-derived CD133+ cells and clones of individual cells were capable of expansion and limited self-renewal and differentiated in vitro into epithelial or endothelial cells. On subcutaneous implantation in SCID mice, the undifferentiated cells formed tubular structures expressing renal epithelial markers. At variance, when differentiated in endothelial cells, these cells formed functional vessels. On intravenous injection in SCID mice with glycerol-induced tubulonecrosis, the in vitro expanded renal-derived CD133+ cells homed into the injured kidney and integrated in tubules. We propose that CD133+ cells from kidney represent a multipotent adult resident stem cell population that may contribute to the repair of renal injury.

Ex vivo expanded endothelial progenitor cells (EPCs) represent a new potential approach for the revascularization of ischemic sites. However, local accumulation of infused EPCs in these sites is poor, and the mechanisms responsible for their homing are largely unknown. We observed the expression of L-selectin, an adhesion receptor that regulates lymphocyte homing and leukocyte rolling and migration, on ex vivo expanded blood-derived human EPCs. When EPCs were subcloned in SV40-T large Ag-transfected isolates, the copresence of L-selectin and endothelial lineage markers was confirmed. We therefore demonstrated that the expression of L-selectin by EPCs was functional because it mediates interaction with a murine endothelial cell line (H.end) expressing L-selectin ligands by way of transfection with alpha(1,3/4)-fucosyltransferase. Indeed, adhesion of EPCs after incubation at 4 degrees C on a rotating platform was enhanced on alpha(1,3/4)-fucosyltransferase-transfected H.end cells compared with control vector-transfected cells, and treatment with anti-L-selectin Abs prevented this event. We then studied the role of L-selectin in EPC homing in vivo. H.end cells were implanted s.c. in SCID mice to form endothelioma tumors, and EPCs were subsequently i.v. injected. L-selectin+ EPCs localized into alpha(1,3/4)-fucosyltransferase-transfected endothelial tumors to a greater extent than in control tumors, and they were able to directly contribute to tumor vascularization by forming L-selectin+ EPC-containing vessels. In conclusion, our results showed that a mechanism typical of leukocyte adhesion is involved in the vascular homing of EPCs within sites of selectin ligand expression. This observation may provide knowledge about the substrate to design strategies to improve EPC localization in damaged tissues.

A simple labeling procedure of stem/progenitor cells based on the use of Gd-HPDO3A and Eu-HPDO3A, respectively, is described. The Gd-chelate acts as T(1)-agent for MRI visualization, whereas the corresponding Eu-chelate acts as reporter in fluorescence microscopy. Owing to their substantial chemical equivalence, the two chelates are equally internalized in EPCs (endothelial progenitor cells), thus allowing their visualization by both techniques. The lanthanide chelates are entrapped in endosomic vesicles and the labeled cells retain biological activity with preservation of viability and pro-angiogenesis capacity. Hyperintense spots in MR have been observed for Gd-labeled EPCs injected under mice kidney capsule or grafted on a subcutaneous Matrigel plug up to 14 days after transplantation., (Copyright 2004 Wiley-Liss, Inc.)

Recent studies demonstrated that factors derived from embryonic stem cells inhibit the tumorigenicity of a variety of cancer cell lines. Embryonic stem cell-secreted Lefty, an inhibitor of Nodal-signalling pathway, was implicated in reprogramming cancer cells. Whether adult stem cells exhibited similar properties has not been explored. The aim of the present study was to investigate whether the conditioned medium (CM) derived from adult stem cells influence in vitro and in vivo tumor growth by a Nodal-dependent pathway. In particular we compared the anti-tumor effect of CM from human liver stem cells (HLSC) with that of bone marrow-derived mesenchymal stem cells (MSC). We found that HLSC-CM inhibited the in vitro growth and promoted apoptosis in HepG2 cells that expressed a deregulated Nodal pathway. The effect of HLSC-CM was related to the presence of Lefty A in the CM of HLSC. Silencing Lefty A in HLSC or Lefty A blockade with a blocking peptide abrogated the anti-proliferative and pro-apoptotic effect of HLSC-CM. Moreover, the administration of human recombinant Lefty A protein mimicked the effect of HLSC-CM indicating that Nodal pathway is critical for the growth of HepG2. At variance of HLSC, bone marrow-derived MSC did not express and release Lefty A and the MSC-CM did not exhibited an anti-tumor activity in vitro, but rather stimulated proliferation of HepG2. In addition, the intra-tumor administration of HLSC-CM was able to inhibit the in vivo growth of HepG2 hepatoma cells implanted subcutaneously in SCID mice. At variance, HLSC-CM derived from Lefty A silenced HLSC was unable to inhibit tumor growth. In conclusion, the results of present study suggest that Lefty A may account for the tumor suppressive activity of HLSC as a result of an inhibition of the Nodal-signalling pathway by a mechanism similar to that described for embryonic stem cells. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSC) were recently shown to migrate to injured tissues when transplanted systemically. The mechanisms underlying the migration and homing of these cells is, however, unclear. In this study, we examine the role of CD44 and its major ligand, hyaluronic acid, in the trafficking of intravenously injected MSC in the glycerol-induced mouse model of acute renal failure (ARF). In vitro, hyaluronic acid promoted a dose-dependent migration of the stem cells that was inhibited by an anti-CD44 blocking monoclonal antibody. In vivo, stem cells injected into mice with ARF migrated to the injured kidney where hyaluronic acid expression was increased. Their presence correlated with morphological and functional recovery. Renal localization of the MSC was blocked by pre-incubation with the CD44 blocking antibody or by soluble hyaluronic acid. Stem cells derived from CD44 knockout mice did not localize to the injured kidney and did not accelerate morphological or functional recovery. Reconstitution by transfection of CD44 knockout stem cells with cDNA encoding wild-type CD44, but not a loss of function CD44 unable to bind hyaluronic acid, restored in vitro migration and in vivo localization of the cells to injured kidneys. We suggest that CD44 and hyaluronic acid interactions recruit exogenous MSC to injured renal tissue and enhance renal regeneration.Kidney International (2007) 72, 430–441; doi:10.1038/sj.ki.5002334; published online 16 May 2007 [ABSTRACT FROM AUTHOR]