BACKGROUND

The epithelial-mesenchymal transition (EMT) is also involved in cancer progression and metastasis. The expression of mesenchymal marker genes might be associated with a more aggressive form of the disease. CD146 is a marker of mesenchymal cells, a potentially metastasis-promoting cell adhesion molecule, and its expression has been described in various types of solid tumors. We, herein, evaluated the expression of CD146 in patients with adenocarcinoma of the lung by using immunohistochemistry to clarify its clinical significance.

METHODS

Tumor specimens were collected from 183 consecutive patients who underwent complete resection for lung adenocarcinoma from 2003 to 2007 in our Department. We analyzed the CD146 expression levels in the primary lung adenocarcinoma by immunohistochemistry.

RESULTS

Positive expression of CD146 was identified in 16 (8.7%) patients. A significant association was only observed between the CD146 expression level and male patient gender (p=0.03); other factors were not associated with CD146 expression. The 5-year overall survival (OS) rate in patients with tumors that were negative and positive for CD146 expression was 84.4% and 50.0%, respectively (p<0.01). A positive CD146 expression was found to be associated with the OS based on a univariate survival analysis (p=0.013).

CONCLUSIONS

CD146 expression was more frequently detected in males than in females. The positive expression of CD146 was associated with a poorer OS according to the survival analysis. CD146 may be a useful marker for predicting poor prognosis in patients with NSCLC following complete resection.

BACKGROUND

Angiogenesis has been suggested as an integral part of inflammatory bowel disease pathology. Vascular endothelial growth factor has long been considered to play a central, specific role in angiogenesis. Endothelial junction adhesion molecules, such as CD146, have recently been suggested to play a potent role in angiogenesis. CD34 is expressed on vascular endothelium, and it has been reported to be upregulated on endothelium in IBD. We investigated the expression of tissue vascular endothelial growth factor, CD34 and CD146 in the inflamed mucosa of patients with active inflammatory bowel disease compared with no inflamed mucosa of healthy controls.

METHODS

Forty-two IBD patients [23 ulcerative colitis, 19 Crohn's disease] and ten healthy controls were included in the study. In colonoscopically obtained biopsies, CD34, CD146 and vascular endothelial growth factor expression were evaluated by immunohistochemistry.

RESULTS

Vascular endothelial growth factor was detected in the mucosa of all groups, and its expression was significantly higher in both Crohn's disease and ulcerative colitis compared with controls (p<0.05). Immunohistochemical staining for CD146 in the inflamed mucosa was significantly higher in both Crohn's disease and ulcerative colitis compared with controls (p=0.002). A trend of higher CD34 expression in Crohn's disease and ulcerative colitis compared with controls was also found, but the difference among the three groups was not statistically significant (p=0.09).

CONCLUSIONS

Inflamed mucosa of patients with active Crohn's disease and ulcerative colitis showed a markedly enhanced expression of VEGF and CD146, than normal mucosa of controls, indicating a possible role of angiogenesis in the pathogenesis of inflammatory bowel disease.

Monoclonal antibody P1H12 recognizes circulating endothelial cells and endothelia of all sizes of blood vessels. To identify the protein recognized by P1H12, we expressed a cDNA library in CHO cells and sequenced the cDNA from positive cells. The P1H12 sequence was identical, except at several bases, to that reported for melanoma cell surface antigen MUC18/CD146. Aggregation assays demonstrated that CD146 mediates Ca(++)-independent homotypic endothelial cell adhesion. P1H12 mAb abrogated interactions between human microvascular endothelial cells (HMVECs) but not between human umbilical vein endothelial cells (HUVECs). P1H12 mAb abrogated P1H12-positive (CHO(P1H12))-association with HMVECs or HUVECs. CD146 distribution is sparser on HUVECs than on HMVECs. These data imply that HMVECs and HUVECs express the CD146 binding partner but that CD146 is functional (or at sufficient density) only on HMVECs. HMVEC monolayers treated with soluble P1H12 mAb showed increased permeability to albumin, with accompanying changes in actin, paxillin, FAK, and caveolin distribution and changes in tyrosine phosphorylation of FAK. Stimulation with P1H12 mAb led to redistribution of NF-kappa B to the nucleus. P1H12 mAb bound to beads inhibited closure of wounded endothelial monolayers. CD146 thus joins VE-cadherin and PECAM-1 as a molecule that mediates homotypic endothelial cell adhesion. CD146 has both structural functions and signaling functions important for endothelial monolayer integrity.

Tissue engineering is an emerging field of science that focuses on creating suitable conditions for the regeneration of tissues. The basic components for tissue engineering involve an interactive triad of scaffolds, signaling molecules, and cells. In this context, stem cells (SCs) present the characteristics of self-renewal and differentiation capacity, which make them promising candidates for tissue engineering. Although they present some common markers, such as cluster of differentiation (CD)105, CD146 and STRO-1, SCs derived from various tissues have different patterns in relation to proliferation, clonogenicity, and differentiation abilities in vitro and in vivo. Tooth-derived tissues have been proposed as an accessible source to obtain SCs with limited morbidity, and various tooth-derived SCs (TDSCs) have been isolated and characterized, such as dental pulp SCs, SCs from human exfoliated deciduous teeth, periodontal ligament SCs, dental follicle progenitor cells, SCs from apical papilla, and periodontal ligament of deciduous teeth SCs. However, heterogeneity among these populations has been observed, and the best method to select the most appropriate TDSCs for regeneration approaches has not yet been established. The objective of this review is to outline the current knowledge concerning the various types of TDSCs, and discuss the perspectives for their use in regenerative approaches.

Bone marrow concentration (BMC) is the most recognized procedure to prepare mesenchymal stem cells for cartilage regeneration. However, bone marrow aspiration is highly invasive and results in low stem cell numbers. Recently, adipose tissue-derived stromal vascular fraction (AT-SVF) was studied as an alternate source of stem cells for cartilage regeneration. However, AT-SVF is not fully characterized in terms of functional equivalence to BMC. Therefore, in this study, we characterized AT-SVF and assessed its suitability as a one-step surgical procedure for cartilage regeneration, as an alternative to BMC. AT-SVF contained approximately sixfold less nucleated cells than BMC. However, adherent cells in AT-SVF were fourfold greater than BMC. Additionally, the colony-forming unit frequency of AT-SVF was higher than that of BMC, at 0.5 and 0.01%, respectively. The mesenchymal stem cell (MSC) population (CD45-CD31-CD90+CD105+) was 4.28% in AT-SVF and 0.42% in BMC, and the adipose-derived stromal cell (ASC) population (CD34+CD31-CD146-) was 32% in AT-SVF and 0.16% in BMC. In vitro chondrogenesis demonstrated that micromass was not formed in BMC, whereas it was clearly formed in AT-SVF. Taken together, uncultured AT-SVF could be used in one-step surgery for cartilage regeneration as a substitute for BMC.

Adoptive cellular immunotherapy has been proposed as an additional treatment of medulloblastoma, an intracranial tumor characterized by a particularly poor prognosis. However, little is known on the ability of the immune system to effectively attack this tumor. In this study, we show that activated human NK cells efficiently kill medulloblastoma cell lines in vitro. NK-mediated killing involved different activating receptors (including NKp46, NKp30, DNAM-1 and NKG2D) and correlated with the presence of their specific ligands on tumor cells. In contrast, the absence of major adhesion interactions, such as LFA-1/ICAM did not impair the NK-mediated cytotoxicity. Medulloblastoma expressed a number of tumor-associated molecules including CD146 and CD133, considered a marker for cancer stem cells. Remarkably, both CD133-positive and CD133-negative cell lines were susceptible to lysis. Tumor cells also expressed molecules that are currently used as diagnostic tools for neuroblastoma cell identification. In particular, B7 homolog 3 (B7-H3) was expressed by all the medulloblastoma cell lines analyzed, while the presence of GD(2) and NB84 was restricted to given cell lines and/or marked a defined tumor cell subset.

BACKGROUND

Cancer stem cells (CSCs) are responsible for treatment failure. However, their identification and roles in resistance are not well established in head and neck squamous cell carcinoma (HNSCC).

METHODS

Three HNSCC cell lines (FaDu, Detroit562 and BICR6) were treated with cisplatin or radiation. Cell surface antigens were analysed by LyoPlate, a novel cell surface antigen array. The expression levels of antigens highly expressed after treatments were further compared between cisplatin-resistant Detroit562 cells and its parental line. Association of the candidate antigen with CSCs properties, namely sphere formation and in vivo tumourigenicity, was also examined.

RESULTS

CD10, CD15s, CD146 and CD282 were upregulated across the treated cell lines, while the increased expression of CD10 was prominent in the cisplatin-resistant cell line. Isolation mediated by FACS revealed that the CD10-positive subpopulation was more refractory to cisplatin, fluorouracil and radiation than the CD10-negative subpopulation. It also showed an increased ability to form spheres in vitro and tumours in vivo. Moreover, the CD10-positive subpopulation expressed the CSC marker OCT3/4 at a higher level than that in the CD10-negative subpopulation.

CONCLUSIONS

CD10 is associated with therapeutic resistance and CSC-like properties of HNSCC. CD10 may serve as a target molecule in the treatment of refractory HNSCC.

Mesenchymal stromal/stem cells (MSCs) reveal progenitor cells-like features including proliferation and differentiation capacities. One of the most historically recognized sources of MSC has been the bone marrow, while other sources recently include adipose tissue, teeth, bone, muscle, placenta, liver, pancreas, umbilical cord, and cord blood. Frequently, progenitor isolation requires traumatic procedures that are poorly feasible and associated with patient discomfort. In the attempt to identify a more approachable MSC source, we focused on endometrial decidual tissue (EDT) found within menstrual blood. Based also on recent literature findings, we hypothesized that EDT may contain heterogeneous populations including some having MSC-like features. Thus, we here sought to isolate EDT-MSC processing menstrual samples from multiple donors. Cytofluorimetric analyses revealed that resulting adherent cells were expressing mesenchymal surface markers, including CD56, CD73, CD90, CD105 and CD146, and pluripotency markers such as SSEA-4. Moreover, EDT-MSC showed a robust clonogenic potential and could be largely expanded in vitro as fibroblastoid elements. In addition, differentiation assays drove these cells towards osteogenic, adipogenic, and chondrogenic lineages. Finally, for the first time, we were able to gene modify these progenitors by a retroviral vector carrying the green fluorescent protein. From these data, we suggest that EDT-MSC could represent a new promising tool having potential within cell and gene therapy applications.

Endothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC-CB.1 and HEPC-CB.2 (human endothelial progenitor cells-cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers: CD133, CD13, CD271, CD90 and also endothelial cell markers: CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells: CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre-angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells.

BACKGROUND

Myofibroblasts play a crucial role in tissue repair. The functional similarities and differences between myofibroblasts and fibroblasts are not fully understood because they have not been separately isolated from a living body. The purpose of this study was to establish a method for the direct isolation of myofibroblasts and fibroblasts from injured lungs by using fluorescence-activated cell sorting and to compare their functions.

RESULTS

We demonstrated that lineage-specific cell surface markers (lin), such as CD31, CD45, CD146, EpCAM (CD326), TER119, and Lyve-1 were not expressed in myofibroblasts or fibroblasts. Fibroblasts of bleomycin-injured lungs and saline-treated lungs were shown to be enriched in linneg Sca-1high, and myofibroblasts of bleomycin-injured lungs were shown to be enriched in linneg Sca-1low CD49ehigh. Results from in-vitro proliferation assays indicated in-vitro proliferation of fibroblasts but not myofibroblasts of bleomycin-injured lungs and of fibroblasts of saline-treated lungs. However, fibroblasts and myofibroblasts might have a low proliferative capacity in vivo. Analysis of genes for collagen and collagen synthesis enzymes by qRT-PCR showed that the expression levels of about half of the genes were significantly higher in fibroblasts and myofibroblasts of bleomycin-injured lungs than in fibroblasts of saline-treated lungs. By contrast, the expression levels of 8 of 11 chemokine genes of myofibroblasts were significantly lower than those of fibroblasts.

CONCLUSIONS

This is the first study showing a direct isolation method of myofibroblasts and fibroblasts from injured lungs. We demonstrated functional similarities and differences between myofibroblasts and fibroblasts in terms of both their proliferative capacity and the expression levels of genes for collagen, collagen synthesis enzymes, and chemokines. Thus, this direct isolation method has great potential for obtaining useful information from myofibroblasts and fibroblasts.

The host immune response to bone biomaterials is vital in determining scaffold fates and bone regeneration outcomes. The nanometer-scale interface of biomaterials, which independently controls physical inputs to cells, regulates osteogenic differentiation of stem cells and local immune response. Herein, we fabricated biomimetic hierarchical intrafibrillarly mineralized collagen (HIMC) with a bone-like staggered nanointerface and investigated its immunomodulatory properties and mesenchymal stem cell (MSC) recruitment during endogenous bone regeneration. The acquired HIMC potently induced neo-bone formation by promoting CD68⁺CD163⁺ M2 macrophage polarization and CD146⁺STRO-1⁺ host MSC recruitment in critical-sized bone defects. Mechanistically, HIMC facilitated M2 macrophage polarization and interleukin (IL)-4 secretion to promote MSC osteogenic differentiation. An anti-IL4 neutralizing antibody significantly reduced M2 macrophage-mediated osteogenic differentiation of MSCs. Moreover, HIMC-loaded-IL-4 implantation into critical-sized mandible defects dramatically enhanced bone regeneration and CD68⁺CD163⁺ M2 macrophage polarization. The depletion of monocyte/macrophages by clodronate liposomes significantly impaired bone regeneration by HIMC, but did not affect MSC recruitment. Thus, in emulating natural design, the hierarchical nanointerface possesses the capacity to recruit host MSCs and promote endogenous bone regeneration by immunomodulation of macrophage polarization through IL-4.

Bone marrow-derived cells have long been regarded to play a crucial role in the homeostasis of skin. We have previously described the clinical benefit of directly applying autologous bone marrow aspirate and cultured bone marrow cells to recalcitrant chronic skin wounds. The initial response to treatment appears to be vascular in nature with the formation of new blood vessels. The difficulty in consistently growing adequate numbers of cells for delivery to patients was, however, a limiting factor. Here, in a subsequent protocol, we describe an improved bone marrow culture system yielding a reliable growth of bone marrow cells and leading to a greater clinical response. Cells expressing markers of endothelial progenitors including CD133, CD146, and particularly CD14 are enhanced in these cultures. CD14-isolated cells produced colonies in endothelial cell assays and sprouting in matrigel assays. Angiogenic cytokines, including angiogenin, epithelial neutrophil-activating protein-78, growth-regulated oncogene, growth-regulated oncogene-alpha, Interleukin-8, CXC16, and monocyte chemoattractant protein-1, were found to be elevated in these cultures. Administration of improved culture cells to patients with chronic wounds present for >1 year lead to an enhanced clinical response.

Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.

Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF.

Dysregulation of Wnt signalling leads to developmental defects and diseases. Non-canonical Wnt signalling via planar cell polarity proteins regulates cell migration and convergent extension; however, the underlying mechanisms are poorly understood. Here we report that Wnt5a uses CD146 as a receptor to regulate cell migration and zebrafish embryonic convergent extension. CD146 binds to Wnt5a with the high affinity required for Wnt5a-induced activation of Dishevelled (Dvl) and c-jun amino-terminal kinase (JNK). The interaction between CD146 and Dvl2 is enhanced on Wnt5a treatment. Mutation of the Dvl2-binding region impairs its ability to activate JNK, promote cell migration and facilitate the formation of cell protrusions. Knockdown of Dvls impairs CD146-induced cell migration. Interestingly, CD146 inhibits canonical Wnt signalling by promoting β-catenin degradation. Our results suggest a model in which CD146 acts as a functional Wnt5a receptor in regulating cell migration and convergent extension, turning off the canonical Wnt signalling branch.

We have previously identified and purified multipotent mesenchymal stromal cell (MSC)-like cells in the highly regenerative endometrial lining of the human uterus (eMSC) as CD140b⁺CD146⁺ cells. Due to ease of accessibility with minimal morbidity via biopsy, we are proposing to use eMSC in cell-based therapies; however, culture conditions compliant with Good Manufacturing Practice have not been established for eMSC. The aim of this study was to optimize serum-free and xeno-free culture conditions for expansion of eMSC for potential clinical use. Real-time cell assessment (Xcelligence) and MTS viability assays were used to measure attachment and proliferation of freshly isolated, flow cytometry-sorted CD140b⁺CD146⁺ eMSC cultured in several commercially available and in-house serum-free and xeno-free media in combination with five attachment matrices (fibronectin, collagen, gelatin, laminin, and Cell Start-XF®). Comparisons were made with a standard serum-containing medium, DMEM/F-12/10% fetal bovine serum. Under all conditions examined, eMSC attachment and proliferation was greatest using a fibronectin matrix, with Lonza TP-SF® and our in-house DMEM/SF/FGF2/EGF serum-free xeno-product-containing medium similar to serum-containing medium. Hypoxia increased eMSC proliferation in the DMEM/SF/FGF2/EGF serum-free medium. Culture of eMSC for 7 days on a fibronectin matrix in DMEM/SF/FGF2/EGF serum-free media in 5% O₂ maintained greater numbers of undifferentiated eMSC expressing CD140b, CD146, and W5C5 compared to culture under similar conditions in Lonza TP-SF medium. However, the percentage of cells expressing typical MSC phenotypic markers, CD29, CD44, CD73, and CD105, were similar for both media. EMSC showed greater expansion in 2D compared to 3D culture on fibronectin-coated microbeads using the optimized DMEM/SF/FGF2/EGF medium in 5% O₂. In the optimized 2D culture conditions, eMSC retained CFU activity, multipotency, and MSC surface phenotype, representing the first steps in their preparation for potential clinical use.

Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culture-derived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of neovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29%+/-7.41%) compared with BM (19.69%+/-5.49%; P=.021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P<.05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P=.119; P=.177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses.

Mesenchymal stem cells (MSC), isolated from dental tissues, are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from human dental pulp (DPSC) of normal impacted third molars that, when cultured in lineage-specific inducing media, differentiate into osteoblasts and adipocytes (evaluated by Alizarin Red S and Red Oil O stainings, respectively), thus showing a multipotency. We confirmed that DPSC, grown under undifferentiating conditions, are negative for hematopoietic (CD45, CD31, CD34, CD144) and positive for mesenchymal (CD29, CD90, CD105, CD166, CD146, STRO-1) markers, that underwent down-regulation when cells were grown in osteogenic medium for 3 weeks. In this condition, they also exhibit an increase in the expression of osteogenic markers (RUNX-2, alkaline phosphatase) and extracellular calcium deposition, whereas the expression of receptors (VEGFR-1 and -2) for vascular endothelial growth factors (VEGF) and related VEGF binding proteins was similar to that found in undifferentiated DPSC. Exposure of DPSC growing under undifferentiating or osteogenic conditions to VEGF-A165 peptide (10-40 ng/ml) for 8 days dose- and time-dependently increased the number of proliferating cells without inducing differentiation towards endothelial lineage, as evaluated by the lack of expression of specific markers (CD31, CD34, CD144). Additionally, exposure of DPSC cultured in osteogenic medium to VEGF-A165 for a similar period enhanced cell differentiation towards osteoblasts as evaluated after 14 and 21 days by Alizarin Red S staining and alkaline phosphatase activity quantification. These findings may have clinical implications possibly facilitating tissue repair and remodeling.

Bone marrow derived mesenchymal stem cells (BM-MSCs) take part in the colonic mucosal regeneration. They are multipotent cells, which can be identified with both negative (i.e. CD13, CD 14, CD45, c-Kit, major histocompatibility complex /MHC class I and II) and positive (i.e. CD54 (ICAM1), CD133, CD146 (MCAM), CD166, Flk-1, Sca-1, Thy-1, stage-specific antigen I /SSEA-I and Musashi-1, HLA class I) markers. These cells can repopulate the gastrointestinal mucosa as they may differentiate into stromal- (i.e. myofi-broblast) or epithelial-like (Paneth-, epithel-, goblet or enteroendocrin) cells without proliferation. During the mesenchymal to epithelial transition (MET) stem cells enter the epithelial layer and take up epithelial cell-like properties. Rarely BM-MSCs may retain their stem cell characteristics and are capable of producing progeny. The isolated lymphoid aggregates may serve as a platform from where BM-MSCs migrate to the nearby crypts as mediated by several chemoattractant proteins, which are expressed in injured tissue. The number of BM-MSCs is influenced by the degree of inflammation. In this review we summarize the current information about the role of BM-MSCs in the repair progress of injured colonic epithelium and their potential clinical applications.

OBJECTIVE

Pediatric solid malignancies display important angiogenic potential, and blocking tumor angiogenesis represents a new therapeutic approach for these patients. Recent studies have evidenced rare circulating cells with endothelial features contributing to tumor neovascularization and have shown the pivotal role of bone marrow-derived (BMD) progenitor cells in metastatic disease progression. We measured these cells in patients with pediatric solid malignancies as a prerequisite to clinical trials with antiangiogenic therapy.

METHODS

Peripheral blood was drawn from 45 patients with localized (n = 23) or metastatic (n = 22) disease, and 20 healthy subjects. Subsets of circulating vascular endothelial growth factor receptor (VEGFR)2+-BMD progenitor cells, defined as CD45-CD34+VEGFR2(KDR)+7AAD- and CD45(dim)CD34+VEGFR2+7AAD- events, were measured in progenitor-enriched fractions by flow cytometry. Mature circulating endothelial cells (CEC) were measured in whole blood as CD31+CD146+CD45-7AAD- viable events. Data were correlated with VEGF and sVEGFR2 plasma levels.

RESULTS

The CD45-CD34+VEGFR2(KDR)+7AAD- subset represented <0.003% of circulating BMD progenitor cells (< or =0.05 cells/mL). However, the median level (range) of the CD45(dim)CD34+VEGFR2+7AAD- subset was higher in patients compared with healthy subjects, 1.5% (0%-10.3%) versus 0.3% (0%-1.6%) of circulating BMD progenitors (P < 0.0001), and differed significantly between patients with localized and metastatic disease, 0.7% (0%-8.6%) versus 2.9% (0.6%-10.3%) of circulating BMD progenitors (P < 0.001). Median CEC value was 7 cells/mL (0-152 cells/mL) and similar in all groups. Unlike VEGFR2+-BMD progenitors, neither CECs, VEGF, or sVEGFR2 plasma levels correlated with disease status.

CONCLUSIONS

High levels of circulating VEGFR2+-BMD progenitor cells correlated with metastatic disease. Our study provides novel insights for angiogenesis mechanisms in pediatric solid malignancies for which antiangiogenic targeting of VEGFR2+-BMD progenitors could be of interest.

Studies on the pathogenesis of osteoporosis and other metabolic bone diseases would be greatly facilitated by the development of approaches to assess changes in gene expression in osteoblast/osteoprogenitor populations in vivo without the potentially confounding effects of in vitro culture and expansion of the cells. While positive selection to identify a progenitor population in human marrow can be used to select for cells capable of osteoblast differentiation, each of the markers that have been used to identify marrow mesenchymal populations (alkaline phosphatase [AP], Stro-1, CD29, CD49a, CD73, CD90, CD105, CD166, CD44, CD146 and CD271) may be expressed on distinct subsets of marrow mesenchymal cells. Thus, positive selection with one or more of these markers could exclude a possibly relevant cell population that may undergo important changes in various clinical conditions. In the present report, we describe the isolation and characterization of human osteoprogenitor cells obtained by depletion of bone marrow cells of all hematopoietic lineage/hematopoietic stem cells and endothelial/endothelial precursor cells (lin-/CD34/CD31-). The yield of lin-/CD34/CD31- cells from ~10 mL of bone marrow (~80 million mononuclear cells) was ~80,000 cells (0.1% of mononuclear cells). While not selected on the basis of expression for the mesenchymal marker, Stro-1, 68% of these cells were Stro-1+. Using linear whole transcriptome amplification followed by quantitative polymerase chain reaction (QPCR) analysis, we also demonstrated that, compared to lin- cells (which are already depleted of hematopoietic cells), lin-/CD34/31- cells expressed markedly lower mRNA levels for the endothelial/hematopoietic markers, CD34, CD31, CD45, and CD133. Lin-/CD34/31- cells were also enriched for the expression of mesenchymal/osteoblastic markers, with a further increase in runx2, osterix, and AP mRNA expression following in vitro culture under osteogenic conditions. Importantly, lin-/CD34/31- cells contained virtually all of the mineralizing cells in human marrow: while these cells displayed robust calcium deposition in vitro, lin-/CD34/31+ cells demonstrated little or no mineralization when cultured under identical osteogenic conditions. Lin-/CD34/31- cells thus represent a human bone marrow population highly enriched for mesenchymal/osteoblast progenitor cells that can be analyzed without in vitro culture in various metabolic bone disorders, including osteoporosis and aging.

Endothelial cells express a wide spectrum of surface molecules involved in multiple vascular functions. We quantitatively determined an extensive immunologic phenotype of endothelial cells through a large panel of antibodies directed against i) well-known endothelial molecules CD31, CD34, CD49b, e, f, CD51, CD54, CD55, CD62E and P, CD105, CD106, HLA class I and HLA class II; ii) molecules defined by monoclonal antibodies newly clustered during the 6th workshop of Human Leukocyte Differentiation Antigen (HLDA) CD109, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146 and CD147; iii) molecules defined by unclustered monoclonal antibodies. The expression of these molecules was quantified on human umbilical vein endothelial cells (HUVEC) cultured in resting conditions and after stimulation with IL-1beta (10 U/ml), TNF-alpha (10 ng/ml) and phorbol myristate acetate (60 ng/ml). Some molecules were constitutively expressed, and others were negative, which served to determine the basal phenotype. After cell stimulation, the molecules showed weak or strong expression modulation, leading to the definition of an activated phenotype. Changes in the kinetics and the amplitude of expression served to characterize poorly defined molecules and may be useful to determine their physiologic role. Also, we compared the phenotypes of endothelial cell lines EA.hy 926 and ECV 304 to that of HUVEC to assess their reliability as an endothelial cell model. Each cell line displayed a specific repertoire of molecules expressed at different levels, which could have significant implications for cell line behavior as endothelial cells.

Gallbladder cancers (GBC) are associated with high disease-specific mortality rates because of no means of early detection and effective therapies. In this study, we investigated CD146 expression, microvessel densities, and lymph vessel densities in 108 adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. We demonstrated that positive CD146 expression, and average microvessel and lymph vessel counts in gallbladder adenocarcinomas were significantly higher than those in peritumoral tissues, polyps, and chronic cholecystitis (ps < 0.01). Positive CD146 expression, and average microvessel and lymph vessel counts were also significantly lower in cases with well-differentiated adenocarcinoma, maximal tumor diameter <2 cm, no metastasis of lymph node, and no invasion of regional tissues than in cases with poorly differentiated adenocarcinoma, maximal tumor diameter ≥ 2 cm, metastasis in lymph nodes, and invasion of regional tissues (p < 0.05 or p < 0.01). Univariate Kaplan-Meier analysis showed that increased expression of CD146 (p = 0.056), higher average microvessel counts (p < 0.05), and lymph vessel counts (p < 0.05) were associated with decreased overall survival. Multivariate Cox regression analysis showed that average microvessel and lymph vessel counts (ps < 0.05) were independent prognostic predictors in gallbladder adenocarcinoma. Our study suggested that the elevated expression of CD146, angiogenesis, and lymphangiogenesis might be closely related to progression, invasion, metastasis, and prognosis of gallbladder adenocarcinoma.

Pluripotent stem cells are still generally accepted not to exist in adult human ovaries, although increasing studies confirm the presence of pluripotent/multipotent stem cells in adult mammalian ovaries, including those of humans. The aim of this study is to isolate, characterize and differentiate in vitro stem cells that originate from the adult human ovarian cortex and that express markers of pluripotency/multipotency. After enzymatic degradation of small ovarian cortex biopsies retrieved from 18 women, ovarian cell cultures were successfully established from 17 and the formation of cell colonies was observed. The presence of cells/colonies expressing some markers of pluripotency (alkaline phosphatase, surface antigen SSEA-4, OCT4, SOX-2, NANOG, LIN28, STELLA), germinal lineage (DDX4/VASA) and multipotency (M-CAM/CD146, Thy-1/CD90, STRO-1) was confirmed by various methods. Stem cells from the cultures, including small round SSEA-4-positive cells with diameters of up to 4 μm, showed a relatively high degree of plasticity. We were able to differentiate them in vitro into various types of somatic cells of all three germ layers. However, these cells did not form teratoma when injected into immunodeficient mice. Our results thus show that ovarian tissue is a potential source of stem cells with a pluripotent/multipotent character for safe application in regenerative medicine.