Circulating tumour cells (CTCs), identified in numerous cancers including melanoma, are unquestionably considered valuable and useful as diagnostic and prognostic markers. They can be detected at all melanoma stages and may persist long after treatment. A crucial step in metastatic processes is the intravascular invasion of neoplastic cells as circulating melanoma cells (CMCs). Only a small percentage of these released cells are efficient and capable of colonizing with a strong metastatic potential. CMCs' ability to survive in circulation express a variety of genes with continuous changes of signal pathways and proteins to escape immune surveillance. This makes it difficult to detect them; therefore, specific isolation, enrichment and characterization of CMC population could be useful to monitor disease status and patient clinical outcome. Overall and disease-free survival have been correlated with the presence of CMCs. Specific melanoma antigens, in particular MCAM (MUC18/MelCAM/CD146), could be a potentially useful tool to isolate CMCs as well as be a prognostic, predictive biomarker. These are the areas reviewed in the article.

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

Microvascular endothelial cells (mECs) circulate at higher numbers in patients with severe sepsis and hemophagocytic syndromes. Although these blood mECs might stem from damaged microvasculature, they are perfectly viable and lead to the establishment of cell lines. Such mECs were cultured in low-dose human serum pools (0.5%) and MEM-alpha medium. Antigenic profiling revealed the expression of CD36, factor VIIIa, CD95-ligand, and CD44, but also CD146. We studied the antioxidative effect of the hematopoietic growth factor G-CSF(1) after in vitro stimulation with LPS from E. coli 0111:B4; the growth factor appeared to exhibit a protective effect on organ function in patients with SIRS. mECs were stimulated with 1 micro g/mL of LPS for 24 h and 48 h with and without G-CSF (3x10(3) U/mL) preincubation. After 24 h, supernatants of the stimulated mEC were tested for IL-8 by ELISA, and cells were tested for hemoxygenase-1 (HO-1, Hsp32) by immunohistochemistry and flow cytometry using OSA110 (mAb, Stressgene). Stimulation with LPS upregulated IL-8 by a factor of 2 to 10 in mEC. Preincubation with G-CSF markedly downregulated the LPS-induced IL-8 secretion (20-50%), but IL-6 production was not affected. Upon 48 h of LPS stimulation, mECs developed massive signs of apoptosis and concomitant caspase 3 activation. Caspase 3 activity induced by LPS (24 h) or by staurosporin (6 h) was found to be dramatically downregulated by the G-CSF preincubation protocol.

The role of angiogenesis as a hallmark of tumor progression has been poorly explored in leiomyosarcoma, a rare but aggressive mesenchymal malignancy. We aimed to characterize microvessel distribution and morphology - including pericyte coverage - in a retrospective series of leyomiosarcomas of the soft tissues and the uterus. 41 whole-block tumor slides from formalin-fixed paraffin-embedded tissues were immunostained for endothelial-specific marker CD31 and microvessel density was quantified by assigning a grade to the frequency of CD31 positive microvessels. Vessel morphology and pericyte coverage were investigated by double-labeling for CD31 and either PDGFRβ, αSMA, desmin, CD90, or CD146. We found that microvessel density correlated with tumor grade in leiomyosarcoma of soft tissues, in analogy with what has been established in several types of carcinoma. This did not apply to uterine leiomyosarcoma, possibly due to the abundant myometrial vascularization. The evaluation of perivascular cell markers related to vessel stability revealed immature microvascular networks with aberrant pericyte coverage, irrespective of tumor origin or grade. Our observations substantiate the role of angiogenesis in the progression of soft tissue leiomyosarcoma. A multiple-marker approach to the assessment of pericyte coverage can identify different profiles of vessel immaturity correlated with tumor grade.

BACKGROUND

Quantification of circulating endothelial cells (CECs) in peripheral blood is developing as a novel and reproducible method of assessing endothelial damage/dysfunction. Accordingly, elevated levels of CECs may be a marker of vascular injury in systemic lupus erythaematosus (SLE). This study was undertaken to assess the blood level of CECs in SLE and to correlate its level with the activity of the disease and to find out the possibility that the presence of increased numbers of CECs can be used as a marker of immune-mediated vessel damage.

METHODS

The study included 33 patients with SLE and 20 healthy controls. They were subjected to clinical examination together with laboratory investigations including complete blood count (CBC), erythrocyte sedimentation rate (ESR), urine analysis, renal function test, C3, C4, ANA, anti-ds DNA antibody, antiphospholipid (IgM and IgG) antibodies and quantification of CECs in blood. CECs were calculated using flow cytometry after staining with a mouse anti-human CD45 antibody (pan-leukocyte marker), mouse anti-human CD146 antibody (endothelial cell marker) and 7-amino-actinomycin D (7-AAD) viability marker. CECs were defined as the live cells with 7-AAD negative, CD45 negative and CD146 positive.

RESULTS

The number of CECs was significantly higher in patients with SLE compared with those in healthy control (mean +/- SD 38.6 +/- 21.2 versus 7.4 +/- 3.4). Furthermore, CECs were correlated positively with SLE disease activity index (SLEDAI) score, ESR and anti-ds DNA. CECs from patients with vasculitic skin lesions, renal and central nervous system (CNS) manifestation were significantly higher than patients free from the previous signs.

CONCLUSIONS

An increased number of CECs observed in patients with SLE was associated with the active phase of the disease and may represent a marker of widespread endothelial injury.

BACKGROUND

Hyperbaric oxygenation was shown to increase bone healing in a rabbit model. However, little is known about the regulatory factors and molecular mechanism involved.We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is mediated via increases in the osteogenic differentiation of mesenchymal stem cells (MSCs) which are regulated by Wnt signaling.

METHODS

The phenotypic characterization of the MSCs was analyzed by flow cytometric analysis. To investigate the effects of HBO on Wnt signaling and osteogenic differentiation of MSCs, mRNA and protein levels of Wnt3a, beta-catenin, GSK-3beta, Runx 2, as well as alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining were analyzed after HBO treatment. To investigate the effects of HBO on Wnt processing and secretion, the expression of Wntless and vacuolar ATPases were quantified after HBO treatment.

RESULTS

Cells expressed MSC markers such as CD105, CD146, and STRO-1. The mRNA and protein levels of Wnt3a, β-catenin, and Runx 2 were up-regulated, while GSK-3β was down-regulated after HBO treatment. Western blot analysis showed an increased β-catenin translocation with a subsequent stimulation of the expression of target genes after HBO treatment. The above observation was confirmed by small interfering (si)RNA treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs. We further showed that HBO treatment increased the expression of Wntless, a retromer trafficking protein, and vacuolar ATPases to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment.

CONCLUSIONS

HBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.

Possible correlations between adiponectin, leptin, CD146, a novel adhesion molecule localized at the endothelial junction, and other markers of endothelial cell injury, von Willebrand factor, thrombomodulin, vascular cell adhesion molecule, and intracellular adhesion molecule, and markers of inflammation, tumor necrosis factor-alpha, interleukin-6, and high-sensitivity C-reactive protein in nondiabetic hemodialyzed patients with and without coronary artery disease were studied. Markers of endothelial dysfunction were elevated in hemodialyzed patients, predominantly with coronary artery disease. In multivariate analysis, kinetic urea modeling and plasminogen activator inhibitor-1 remained the only positive predictors of adiponectin. In multivariate analysis, predictors of leptin were triglycerides, tissue plasminogen activator, CD146, and coronary artery disease. In multivariate analysis, predictors of CD146 were age, hemoglobin, and adiponectin. Elevated adiponectin correlated to CD146 may be the expression of a counterregulatory response aimed at mitigating the consequences in endothelial damage and increased cardiovascular risk in renal failure. The data provide further support for a link between adipocytokines, endothelial dysfunction, cardiovascular risk, and renal failure.

OBJECTIVE

Stem cells from human exfoliated deciduous teeth (SHED) are a good source of dental tissue for regeneration therapy, and can be obtained using different primary culture methods. The aim of this study was to determine the differences in the in vitro and in vivo characteristics between SHED isolated via enzymatic disaggregation (e-SHED) and outgrowth (o-SHED) primary culture methods.

METHODS

Dental pulp stem cells were isolated from 14 exfoliated deciduous teeth by enzymatic disaggregation (n=7) and outgrowth (n=7). Their proliferation potential and colony-forming ability were evaluated in vitro, as was their mesenchymal stem-cell-marker expression (using flow cytometry), and their differentiation was verified using quantitative real-time PCR (qPCR) and histochemical staining. In addition, the qualitative and quantitative characteristics of the hard tissue that was generated after in vivo transplantation were compared using haematoxylin and eosin staining, immunohistochemical staining, qPCR, and quantitative alkaline phosphatase analysis.

RESULTS

The cell-proliferation potential, colony-forming ability, and Stro-1 and CD146 expression were higher in e-SHED than in o-SHED. While the in vitro adipogenic differentiation potential was greater in e-SHED than in o-SHED, the in vitro osteogenic differentiation did not differ significantly between the two cell types. Although in vivo hard tissue formation was greater following transplantation of o-SHED into mice, there was no difference in the quality of hard tissue generated by e-SHED and o-SHED.

CONCLUSIONS

The findings of this study indicate that e-SHED exhibit stronger stemness characteristics, but that o-SHED are more suitable for hard-tissue regeneration therapy in teeth.

OBJECTIVE

Metastasis is a complex process involving multiple genetic changes. The high mortality and poor prognosis caused by metastasis in malignant tumor patients and the uncertain mechanisms are always the prominent problems in the field of oncology. In order to screen for lymphatic metastasis-associated genes, the gene expression profiles of mouse hepatocarcinoma cell lines Hca-F (highly metastatic) and Hca-P (low metastatic) were compared by gene chip.

METHODS

Total RNA was isolated from Hca-F and Hca-P cells, and synthesized into double-stranded cDNA, then synthesized into biotin-labeled cRNA probes by in vitro transcription. The cRNA probes were separately hybridized with Affymetrix GeneChip MOE430A (containing 22,690 transcripts, including 14,500 known mouse genes and 4,371 ESTs), and the signals were scanned by the GeneArray Scanner. The results were analyzed by bioinformatics.

RESULTS

Compared with the gene expression profile of Hca-P cells, 901 (6.2%) genes and 129 (3%) ESTs were up-regulated by at least 2 folds in Hca-F cells; 33 genes, including endoglin (EDG; CD105), Mcam (Muc18; Mel-CAM; CD146), Cdc42ep5 (CEP5; Borg3), Ptprr (protein tyrosine phosphatase, receptor type, R), F2r [coagulation factor II (thrombin) receptor; Par1; ThrR], D7Ertd458e (necl-5), NR1D1, Serpin h1 (HSP47), AXL, Mak, and Areg (AR), were up-regulated 13.93-29.86 folds. According to Gene Ontology and Treeview analysis, these 33 genes were involved in angiogenesis, cell adhesion, signal transduction, cell motility, transcription, chaperone activity, protein kinase activity, receptor binding, and so on.

CONCLUSIONS

Many lymphatic metastasis-associated genes were screened by high-throughput gene chip method; validating their cellular functions will help to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as diagnostic markers and therapeutic targets for lymphatic metastasis.

BACKGROUND

Recent studies proposed an increased risk of atherosclerosis in patients with a history of Kawasaki disease. This study aimed to investigate the persistence of vascular injury after an acute phase of the Kawasaki disease.

METHODS

We determined the number of circulating endothelial cells (CEC) in the peripheral blood of 13 patients with a history of Kawasaki disease within four to ten years, in comparison with 13 healthy relative controls. The CECs were counted as CD146+/CD34 + cells by the standard flow cytometry technique, and the independent t-test was employed to compare the mean number of CECs in the two groups.

RESULTS

The mean number of CECs was significantly higher in patients than in controls (12 ± 3.03 vs. 2.38 ± 0.87, respectively, P < 0.001).

CONCLUSIONS

This study elucidates the persistence of vascular injury late after Kawasaki disease. This finding suggests that prolonged administration of vascular anti-inflammatory agents might be beneficial for preventing atherosclerosis in the subsequent years, in these patients.

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.

BACKGROUND

Uric acid may play a pathogenic role in hypertension, cardiovascular morbidity, and kidney disease. The aim of this study was to assess the effect of serum uric acid on biomarkers of endothelial activation and renal function in kidney allograft recipients during 30 months of follow-up.

METHODS

The study included 100 allograft recipients with stable renal function (estimated glomerular filtration rate (eGFR) >60 mL/min). The study was performed 34 ± 12 months after transplantation. The patients were followed prospectively for 30 months. Seventy patients displayed hyperuricemia (uric acid 7.5 ± 1.0 mg/dL) and 30 normouricemia (5.5 ± 0.9 mg/dL). Concentrations of plasma resistin, soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble CD146, and high-sensitivity C-reactive protein (hs-CRP) were assessed in patients at the beginning and after 30 months of follow-up. Clinical outcomes and biomarker values were analyzed in these groups and compared to a control group of 26 healthy volunteers.

RESULTS

Concentrations of resistin and CD146 were increased among the hyperuricemia versus the normouricemic group (P < .05). Serum uric acid level correlated with sVCAM-1, hs-CRP, resistin, and sCD146 concentration in both groups of kidney recipients (P < .01). Serum creatinine concentrations correlated with sVCAM-1, resistin, and sCD146 concentrations (P < .01). There were significant direct correlations between uric acid and the number of antihypertensive agents (P < .001) and inverse correlations between eGFR (P < .001) and high-density lipoprotein cholesterol (P < .04). Pulse pressure increased in hyperuricemic patients during follow-up (P < .05). The decrease in eGFR during the 30-month follow-up was similar in both groups. No subject progressed to kidney allograft failure. Patient and graft survivals were 98% among hyperuricemic and 96.7% among normouricemic individuals.

CONCLUSIONS

Hyperuricemia may injure endothelial function via resistin-dependent mechanisms. It represents a risk factor for arterial stiffness. The elevated serum uric acid may not have a causal role in the progression of renal transplant injury over 30 months of follow-up.

BACKGROUND

The ferret canine tooth has been introduced as a suitable model for studying dental pulp regeneration. The aim of this study was to isolate and characterize ferret dental pulp stem cells (fDPSCs) and their differentiation potential.

METHODS

Dental pulp stem cells were isolated from freshly extracted ferret canine teeth. The cells were examined for the expression of stem cell markers STRO-1, CD90, CD105, and CD146. The osteo/odontogenic and adipogenic differentiation potential of fDPSCs was evaluated. Osteogenic and odontogenic marker genes were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) on days 1, 4, and 8 after osteo/odontogenic induction of fDPSCs including dentin sialophosphoprotein (DSPP), dentin matrix protein-1, osteopontin, and alkaline phosphatase. Human dental pulp cells were used as the control. The results were analyzed using 3-way analysis of variance.

RESULTS

fDPSCs were positive for STRO1, CD90, and CD105 and negative for CD146 markers with immunohistochemistry. fDPSCs showed strong osteogenic and weak adipogenic potential. The overall expression of DSPP was not significantly different between fDPSCs and human dental pulp cells. The expression of DSPP in osteo/odontogenic media was significantly higher in fDPSCs on day 4 (P < .01). The overall expression of dentin matrix protein-1, osteopontin, and alkaline phosphatase was significantly higher in fDPSCs (P = .0005).

CONCLUSIONS

fDPSCs were positive for several markers of dental pulp stem cells resembling human DPSCs and appeared to show a stronger potential to differentiate to osteoblastic rather than odontoblastic lineage.

Diabetes damages the endothelium and reduces the availability of bone marrow (BM)-derived endothelial progenitor cells (EPCs). The mobilization of hematopoietic stem cells (HSCs) and EPCs in response to G-CSF is impaired by diabetes, owing to CXCL12 dysregulation. We have previously shown that the CXCR4/CXCL12 disruptor plerixafor rescues HSC and EPC mobilization in diabetes. We herein explored the effects of plerixafor on HSCs, EPCs, and circulating endothelial cells (CECs) in patients with and without diabetes.

We re-analysed data gathered in the NCT02056210 trial, wherein patients with (n = 10) and without diabetes (n = 10) received plerixafor to test stem/progenitor cell mobilization. We applied a novel and very specific polychromatic flow cytometry (PFC) approach to identify and quantify HSCs, EPCs, and CECs.

We found that 7-AAD(-)Syto16(+)CD34(+)CD45(dim) HSC levels determined by PFC strongly correlated to the traditional enumeration of CD34(+) cells, whereas 7-AAD(-)Syto16(+)CD34(+)CD45(neg)KDR(+) EPCs were unrelated to the traditional enumeration of CD34(+)KDR(+) cells. Using PFC, we confirmed that plerixafor induces rapid mobilization of HSCs and EPCs in both groups, with a marginally significant defect in patients with diabetes. Plerixafor reduced live (7-AAD(-)) and dead (7-AAD(+)) Syto16(+)CD34(bright)CD45(neg)CD146(+) CECs more in patients without than in those with diabetes. The EPC/CEC ratio, a measure of the vascular health balance, was increased by plerixafor, but less prominently in patients with that in those without diabetes.

In addition to rescuing defective mobilization associated with diabetes, plerixafor improves the balance between EPCs and CECs, but the latter effect is blunted in patients with diabetes.

The advantages of adipose-derived stem cells (AdSCs) over bone marrow stem cells (BMSCs), such as being available as a medical waste and less discomfort during harvest, have made them a good alternative instead of BMSCs in tissue engineering. AdSCs from buccal fat pad (BFP), as an easily harvestable and accessible source, have gained interest to be used for bone regeneration in the maxillofacial region. Due to scarcity of data regarding comparative analysis of isolated AdSCs from different parts of the body, we aimed to quantitatively compare the proliferation and osteogenic capabilities of AdSCs from different harvesting sites. In this study, AdSCs were isolated from BFP (BFPdSCs), abdomen (abdomen-derived mesenchymal stem cells (AbdSCs)), and hip (hip-derived mesenchymal stem cells (HdSCs)) from one individual and were compared for surface marker expression, morphology, growth rate, and osteogenic differentiation capability. Among them, BFPdSCs demonstrated the highest proliferation rate with the shortest doubling time and also expressed vascular endothelial markers including CD34 and CD146. Moreover, the expression of osteogenic markers were significantly higher in BFPdSCs. The results of this study suggested that BFPdSCs as an encouraging source of mesenchymal stem cells are to be used for bone tissue engineering.

The therapeutic applications of mesenchymal stem cells (MSCs) have been actively explored due to their broad anti-inflammatory and immunomodulatory properties. However, the use of xenogeneic components, including fetal bovine serum (FBS), in the expansion media might pose a risk of xenoimmunization and zoonotic transmission to post-transplanted patients. Here, we extensively compared the physiological functions of human Wharton's jelly-derived MSCs (WJ-MSCs) in a xeno-free medium (XF-MSCs) and a medium containing 10% FBS (10%-MSCs). Both groups showed similar proliferation potential; however, the 10%-MSCs showed prolonged expression of CD146, with higher colony-forming unit-fibroblast (CFU-F) ability than the XF-MSCs. The XF-MSCs showed enhanced adipogenic differentiation potential and sufficient hematopoietic stem cell (HSC) niche activity, with elevated niche-related markers including CXCL12. Furthermore, we demonstrated that the XF-MSCs had a significantly higher suppressive effect on human peripheral blood-derived T cell proliferation, Th1 and Th17 differentiation, as well as naïve macrophage polarization toward an M1 phenotype. Among the anti-inflammatory molecules, the production of indoleamine 2,3-dioxygenase (IDO) and nitric oxide synthase 2 (NOS2) was profoundly increased, whereas cyclooxygenase-2 (COX-2) was decreased in the XF-MSCs. Finally, the XF-MSCs had an enhanced therapeutic effect against mouse experimental colitis. These findings indicate that xeno-free culture conditions improved the immunomodulatory properties of WJ-MSCs and ex vivo-expanded XF-MSCs might be an effective strategy for preventing the progression of colitis.

Keywords: colitis model; immunomodulatory property; indoleamine 2,3-dioxygenase (IDO); mesenchymal stem cells; stem cell therapy; xeno-free medium.

Mesenchymal stromal cells (MSCs) are the most common cell population studied for therapeutic use in veterinary medicine. MSCs obtained from neonatal sources such as umbilical cord tissue (CT-MSCs) or cord blood (CB-MSCs) are appealing due to the non-invasive nature of procurement and the time allowed for characterization of cells prior to use. However, it remains unclear whether CB- or CT-MSCs have equivalent progenitor and non-progenitor functions. CB-MSCs have been shown to have superior chondrogenic potential to MSCs from other sources, whereas their immunomodulatory capacity does not seem to vary significantly. Using equine CB-MSCs and CT-MSCs from the same donor, we hypothesized that MSCs from both sources would have a similar immunophenotype, that CB-MSCs would be more amenable to differentiation, and that they can equally suppress lymphocyte proliferation. We evaluated cells from both sources for "classic" equine MSC markers CD90, CD105, CD29, and CD44, as well as pericyte markers CD146, NG2 and α-SMA. Contrary to our hypothesis, CB-MSCs showed mid- to high expression of pericyte surface markers CD146 and NG2, while expression in CT-MSCs was absent. Upon trilineage differentiation, CB-MSCs were more osteogenic and chondrogenic based on ALP activity and glycosaminoglycan content, respectively. Finally, using a mononuclear cell suppression assay, we determined that CB-MSCs and CT-MSCs are both capable of suppressing stimulated mononuclear cell proliferation to a similar degree. We have determined that choice of MSC tissue source should be made with the intended application in mind. This appears to be particularly relevant if pursuing a progenitor-based treatment strategy.

: Engineered and devitalized hypertrophic cartilage (HC) has been proposed as bone substitute material, potentially combining the features of osteoinductivity, resistance to hypoxia, capacity to attract blood vessels, and customization potential for specific indications. However, in comparison with vital tissues, devitalized HC grafts have reduced efficiency of bone formation and longer remodeling times. We tested the hypothesis that freshly harvested stromal vascular fraction (SVF) cells from human adipose tissue-which include mesenchymal, endothelial, and osteoclastic progenitors-enhance devitalized HC remodeling into bone tissue. Human SVF cells isolated from abdominal lipoaspirates were characterized cytofluorimetrically. HC pellets, previously generated by human bone marrow-derived stromal cells and devitalized by freeze/thaw, were embedded in fibrin gel with or without different amounts of SVF cells and implanted either ectopically in nude mice or in 4-mm-diameter calvarial defects in nude rats. In the ectopic model, SVF cells added to devitalized HC directly contributed to endothelial, osteoblastic, and osteoclastic populations. After 12 weeks, the extent of graft vascularization and amount of bone formation increased in a cell-number-dependent fashion (up to, respectively, 2.0-fold and 2.9-fold using 12 million cells per milliliter of gel). Mineralized tissue volume correlated with the number of implanted, SVF-derived endothelial cells (CD31+ CD34+ CD146+). In the calvarial model, SVF activation of HC using 12 million cells per milliliter of gel induced efficient merging among implanted pellets and strongly enhanced (7.3-fold) de novo bone tissue formation within the defects. Our findings outline a bone augmentation strategy based on off-the-shelf devitalized allogeneic HC, intraoperatively activated with autologous SVF cells.

This study validates an innovative bone substitute material based on allogeneic hypertrophic cartilage that is engineered, devitalized, stored, and clinically used, together with autologous cells, intraoperatively derived from a lipoaspirate. The strategy was tested using human cells in an ectopic model and an orthotopic implantation model, in immunocompromised animals.

Head and neck cancer is one of the most common cancers in Europe. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells, including adult stem cells. One of the fundamental properties of an adult stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. However, under certain stimuli, unspecialized adult stem cells can give rise to specialized cells to generate replacements for cells that are lost during one's life or due to injury or disease. Nevertheless, specialization of stem cells must be controlled by specific milieu and also initiated at the proper time, making the entire process beneficial for tissue recovery and maintaining it for a long time. In this paper we assess whether irradiated dental pulp stem cells have maintained open their options to mature into specialized cells, or whether they have lost their unspecialized (immature) state following irradiation. Our findings showed radiation-induced premature differentiation of dental pulp stem cells towards odonto-/osteoblast lineages in vitro. Matrix calcification was visualized from Day 6 or Day 9 following irradiation of cells expressing low or high levels of CD146, respectively.

The role of angiogenesis in multiple myeloma (MM) pathogenesis is well established. Angiogenesis is linked to the functional state of endothelial junctions that are modulated by the growth and activation of endothelial cells. CD146 and vascular endothelial-cadherin (VE-cadherin) are cell adhesion molecules localized at the endothelial junction. The aim of the study was to assess sVE-cadherin and sCD146 serum levels in MM patients. Forty-six untreated patients with MM were included in this study. In addition, 23 of 46 patients were analyzed again in partial remission after initial chemotherapy. Twenty-two samples from healthy volunteers were evaluated as the control. There was no significant difference in sCD146 level between MM patients and the control (511 +/- 177.2 vs. 460.9 +/- 156.9 ng/ml respectively). In untreated MM patients, sVE-cadherin level was significantly higher than in the control (1.36 +/- 0.55 vs. 0.63 +/- 0.56 ng/ml respectively; P < 0.05). In untreated MM patients, sVE-cadherin level was significantly higher than in MM patients in partial remission (1.36 +/- 0.55 vs. 0.5 +/- 0.33 respectively; P < 0.05). sVE-cadherin but not sCD146 serum level was increased in untreated MM patients and decreases after chemotherapy in patients in partial remission. VE-cadherin may reflect intensity of angiogenesis in MM and may be useful in prognosis of response to treatment.

Bronchopulmonary dysplasia (BPD), the most common complication of extreme preterm birth, can be caused by oxygen-related lung injury and is characterized by impaired alveolar and vascular development. Mesenchymal stromal cells (MSCs) have lung protective effects. Conversely, BPD is associated with increased MSCs in tracheal aspirates. We hypothesized that endogenous lung (L-)MSCs are perturbed in a well-established oxygen-induced rat model mimicking BPD features. Rat pups were exposed to 21% or 95% oxygen from birth to postnatal day 10. On day 12, CD146+ L-MSCs were isolated and characterized according to the International Society for Cellular Therapy criteria. Epithelial and vascular repair potential were tested by scratch assay and endothelial network formation, respectively, immune function by mixed lymphocyte reaction assay. Microarray analysis was performed using the Affymetrix GeneChip and gene set enrichment analysis software. CD146+ L-MSCs isolated from rat pups exposed to hyperoxia had decreased CD73 expression and inhibited lung endothelial network formation. CD146+ L-MSCs indiscriminately promoted epithelial wound healing and limited T cell proliferation. Expression of potent antiangiogenic genes of the axonal guidance cue and CDC42 pathways was increased after in vivo hyperoxia, whereas genes of the anti-inflammatory Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and lung/vascular growth-promoting fibroblast growth factor (FGF) pathways were decreased. In conclusion, in vivo hyperoxia exposure alters the proangiogenic effects and FGF expression of L-MSCs. In addition, decreased CD73 and JAK/STAT expression suggests decreased immune function. L-MSC function may be perturbed and contribute to BPD pathogenesis. These findings may lead to improvements in manufacturing exogenous MSCs with superior repair capabilities.

Pericytes are important cellular components of the tumor microenviroment with established roles in angiogenesis and metastasis. These two cancer hallmarks are modulated by enzymes of the LOX family, but thus far, information about LOX relevance in tumor-associated pericytes is lacking. Here, we performed a comparative characterization of normal and tumoral pericytes and report for the first time the modulatory effects of LOX enzymes on activated pericyte properties. Tumoral pericytes isolated from childhood ependymoma and neuroblastoma specimens displayed angiogenic properties in vitro and expressed typical markers, including CD146, NG2, and PDGFRβ. Expression of all LOX family members could be detected in both normal and tumor-associated pericytes. In most pericyte samples, LOXL3 was the family member displaying the highest transcript levels. Inhibition of LOX/LOXL activity with the inhibitor β-aminopropionitrile (βAPN) significantly reduced migration of pericytes, while proliferation rates were kept unaltered. Formation of tube-like structures in vitro by pericytes was also significantly impaired upon inhibition of LOX/LOXL activity with βAPN, which induced more prominent effects in tumor-associated pericytes. These findings reveal a novel involvement of the LOX family of enzymes in migration and angiogenic properties of pericytes, with implications in tumor development and in therapeutic targeting tumor microenvironment constituents.

Both vasculogenesis and angiogenesis occur during normal placental vascular development. Additionally, the placenta undergoes a process of vascular mimicry (pseudo-vasculogenesis) where the placental extravillous trophoblast (EVT) that invade the spiral arteries convert from an epithelial to an endothelial phenotype during normal pregnancy. As soluble CD146 (sCD146) constitutes a new physiological factor with angiogenic properties, we hypothesized that it could be involved in the regulation of placental vascular development by acting on EVT. Using placental villous explants, we demonstrated that sCD146 inhibits EVT outgrowth. Consistently, we showed that sCD146 inhibits the ability of EVT cells (HTR8/SVneo) to migrate, invade and form tubes in Matrigel, without affecting their proliferation or apoptosis. The involvement of sCD146 in human pregnancy was investigated by evaluation of sCD146 levels in 50 pregnant women. We observed physiological down-regulation of sCD146 throughout pregnancy. These results prompted us to investigate the effect of prolonged sCD146 administration in a rat model of pregnancy. Repeated systemic sCD146 injections after coupling caused a significant decrease of pregnancy rate and number of embryos. Histological studies performed on placenta evidenced a reduced migration of glycogen cells (analogous to EVT in rat) in sCD146-treated rats. We propose that in human, sCD146 could represent both an attractive biomarker of placental vascular development and a therapeutic target in pregnancy complications associated with pathological angiogenesis.