Adipose tissue contains abundant multipotent mesenchymal stem cells with strong proliferative and differentiating potential into adipocytes, osteocytes, and chondrocytes. However, adipose-derived mesenchymal stem cells (ASCs) showed variable characteristics based on the tissue-harvesting site. This study aimed at comparing human adipose-derived mesenchymal stem cell from the orbit (Orbital ASCs) and abdomen (Abdominal ASCs). Orbital and abdominal ASCs were isolated during an upper or lower blepharoplasty operation and liposuction, respectively. Flow cytometric analysis was done to analyze the surface antigens of ASCs, and cytokine profiles were measured using Luminex assay kit. The multilineage potential of both ASCs was investigated using Oil Red O, alizarin red, and alcian staining. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to measure mRNA levels of genes involved in these trilineage differentiations. Our results showed that both types of ASCs expressed the cell surface markers which are commonly expressed stem cells; however, orbital-ASCs showed higher expressions of CD73, CD90, CD105, and CD146 than abdominal ASCs. Unlikely, orbital-ASC expressed CD31, CD45 and HLA-DR lesser than abdominal-ASCs. Orbital ASCs secreted higher concentrations of eotaxin, fractalkine, IP-10, GRO, MCP-1, IL-6, IL-8, and RANTES but lower MIP-1α, FGF-2, and VEGF concentrations than abdominal-ASCs. Our result showed that orbital ASCs have higher potential towards adipogenic and osteogenic differentiation but lower tendency to chondrogenesis when compared with abdominal ASCs. In conclusion, tissue-harvesting site is a strong determinant for characterization of adipose-derived mesenchymal stem cells. Understanding defining phenotypes of such cells is useful for making suitable choices in different regenerative clinical indications.

The circulating endothelial progenitor cells (EPCs) in blood of acute myocardial infarction (AMI) patient have been monitored in many previous studies. The number of circulating EPC increases in the blood of patients at onset of the AMI. EPC is originated from bone marrow. It performs vessel regeneration. There are many markers used for detecting EPC. Four of these markers, CD31, CD34, CD45, and CD146, were concurrently detected at the single cell level for the identification of EPC in the present preliminary study. The CD45 negative cell sorting was performed to peripheral blood mononuclear cells (PBMCs) acquired from four AMI patients with a magnetic bead sorter, since, EPCs expressed CD45 negative or dim. The resultant PBMC eluents were treated with quantum-antibody conjugates for the probing four different markers of EPCs and then applied to a high-content single cell imaging cytometer using acousto-optical tunable filter (AOTF). The use of quantum dot, with narrow emission wavelength range and AOTF enabling cellular image at a particular single wavelength, is very advantageous for accurate high-content AMI diagnosis based on simultaneous monitoring of many markers. The number of EPC increased as compared with control in three of four AMI patients. In this approach, two EPC subtypes were found, CD31(+), CD34(+), CD45(-/dim), CD146(-) as early outgrowth EPCs and CD31(+), CD34(+), CD45(-/dim), CD146(+) as late outgrowth EPCs. Patient 1 had CD31(+), CD34(+), CD45(-/dim), CD146(+) cells whose percentage was 4.21% of cells. Patient 2 had 2.38% of CD31(+), CD34(+), CD45(-/dim), CD146(-) cells and patient 3 had 4.28% of CD31(+), CD34(+), CD45(-/dim), CD146(+) cells.

Over the last decade and half, the optimization of cryopreservation for adipose tissue derived stromal/stem cells (ASCs) especially in determining the optimal combination of cryoprotectant type, cooling rate, and thawing rate have been extensively studied. In this study, we examined the functionality of ASCs that have been frozen-stored for more than 10 years denoted as long-term freezing, frozen within the last 3 to 7 years denoted as short-term freezing and compared their response with fresh ASCs. The mean post-thaw viability for long-term frozen group was 78% whereas for short-term frozen group 79% with no significant differences between the two groups. The flow cytometry evaluation of stromal surface markers, CD29, CD90, CD105, CD44, and CD73 indicated the expression (above 95%) in passages P1-P4 in all of the frozen-thawed ASC groups and fresh ASCs whereas the hematopoietic markers CD31, CD34, CD45, and CD146 were expressed extremely low (below 2%) within both the frozen-thawed and fresh cell groups. Quantitative real time polymerase chain reaction (qPCR) analysis revealed some differences between the osteogenic gene expression of long-term frozen group in comparison to fresh ASCs. Intriguingly, one group of cells from the short-term frozen group exhibited remarkably higher expression of osteogenic genes in comparison to fresh ASCs. The adipogenic differentiation potential remained virtually unchanged between all of the frozen-thawed groups and the fresh ASCs. Long-term cryopreservation of ASCs, in general, has a somewhat negative impact on the osteogenic potential of ASCs, especially as it relates to the decrease in osteopontin gene expression but not significantly so with respect to RUNX2 and osteonectin gene expressions. However, the adipogenic potential, post thaw viability, and immunophenotype characteristics remain relatively intact between all the groups.

OBJECTIVE

Nano dense-silica (dSiO2) has many advantages such as adjustable core-shell structure, multiple drug delivery, and controllable release behavior. Improving the gastric tumor-specific targeting efficiency based on the development of various strategies is crucial for anti-cancer drug delivery systems.

METHODS

Superparamagnetic iron oxide nanoparticles (SPION) were coated with dSiO2 as core-shell nanoparticles, and labeled with near infra-red fluorescence (NIRF) dye 800ZW (excitation wavelength: 778 nm/emission wavelength: 806 nm) and anti-CD146 monoclonal antibody YY146 for magnetic resonance (MR)/NIRF imaging study in xenograft gastric cancer model. The morphology and the size of pre- and postlabeling SPION@dSiO2 core-shell nanoparticles were characterized using transmission electron microscopy. Iron content in SPION@dSiO2 nanoparticles was measured by inductively coupled plasma optical emission spectrometry. Fluorescence microscopy and fluorescence-activated cell sorter studies were carried out to confirm the binding specificity of YY146 and 800ZW-SPION@dSiO2-YY146 on MKN45 cells. In vivo and in vitro NIRF imaging, control (nanoparticles only) and blocking studies, and histology were executed on MKN45 tumor-bearing nude mice to estimate the affinity of 800ZW-SPION@dSiO2-YY146 to target tumor CD146.

RESULTS

800ZW-SPION@dSiO2-YY146 nanoparticles were uniformly spherical in shape and dispersed evenly in a cell culture medium. The diameter of the nanoparticle was 20-30 nm with 15 nm SPION core and ~10 nm SiO2 shell, and the final concentration was 1.7 nmol/mL. Transverse relaxivity of SPION@dSiO2 dispersed in water was measured to be 110.57 mM(-1)·s(-1). Fluorescence activated cell sorter analysis of the nanoparticles in MKN45 cells showed 14-fold binding of 800ZW-SPION@dSiO2-YY146 more than the control group 800ZW-SPION@dSiO2. Series of NIRF imaging post intravenous injection of 800ZW-SPION@dSiO2-YY146 demonstrated that the MKN45 xenograft tumor model could be clearly identified as early as a time point of 30 minutes postinjection. Quantitative analysis revealed that the tumor uptake peaked at 24 hours postinjection.

CONCLUSIONS

This is the first successful study of functional nanoparticles for MR/NIRF imaging of cell surface glycoprotein CD146 in gastric cancer model. Our results suggest that 800ZW-SPION@dSiO2-YY146 nanoparticles will be applicable in tumor for image-guided therapy/surgery.

Tumor-propagating cells (TPCs) are believed to drive cancer initiation, progression and recurrence. These cells are characterized by enhanced tumorigenicity and self-renewal. The ability to identify such cells in primary human sarcomas relies on the dye exclusion ability of tumor side population (SP) cells. Here, we performed a high-throughput cell surface antigen screen and found that CD146 is enriched in the SP population. In vivo serial transplantation assays showed that CD146+ cells are highly tumorigenic, capable of self-renewal and thus enriches for the TPC population. In addition, depletion of SP cells from the CD146+ population show that CD146+ cells and SP cells are a distinct and overlapping TPC populations. Gene expression profiling of CD146+ and SP cells revealed multiple pathways commonly upregulated in both of these populations. Inhibition of one of these upregulated pathways, Notch signaling, significantly reduced tumor growth and self-renewal. Our data demonstrate that CD146 is an effective cell surface marker for enriching TPCs in primary human sarcomas. Targeting differentially activated pathways in TPCs may provide new therapeutic strategies for treating sarcoma.

Stromal cells/telocytes (SCs/TCs) were recently described in the human adult trigeminal ganglion (TG). As some markers are equally expressed in SCs/TCs and endothelial cells, we hypothesized that a subset of the TG SCs/TCs is in fact represented by endothelial progenitor cells of a myelomonocytic origin. This study aimed to evaluate whether the interstitial cells of the human adult TG correlate with the myelomonocytic lineage. We used primary antibodies for c-erbB2/HER-2, CD31, nestin, CD10, CD117/c-kit, von Willebrand factor (vWF), CD34, Stro-1, CD146, α-smooth muscle actin (α-SMA), CD68, VEGFR-2 and cytokeratin 7 (CK7). The TG pial mesothelium and subpial vascular microstroma expressed c-erbB2/HER-2, CK7 and VEGFR-2. SCs/TCs neighbouring the neuronoglial units (NGUs) also expressed HER-2, which suggests a pial origin. These cells were also positive for CD10, CD31, CD34, CD68 and nestin. Endothelial cells expressed CD10, CD31, CD34, CD146, nestin and vWF. We also found vasculogenic networks with spindle-shaped and stellate endothelial progenitors expressing CD10, CD31, CD34, CD68, CD146 and VEGFR-2. Isolated mesenchymal stromal cells expressed Stro-1, CD146, CK7, c-kit and nestin. Pericytes expressed α-SMA and CD146. Using transmission electron microscopy (TEM), we found endothelial-specific Weibel-Palade bodies in spindle-shaped stromal progenitors. Our study supports the hypothesis that an intrinsic vasculogenic niche potentially involved in microvascular maintenance and repair might be present in the human adult trigeminal ganglion and that it might be supplied by either the pial mesothelium or the bone marrow niche.

Increased risk of cardiovascular disease in end-stage renal disease (ESRD) has been explained by accelerated atherosclerosis and impaired angiogenesis, in which endothelial progenitor cells (EPC) may play key roles. Circulating cells with endothelial progenitor phenotype have not been evaluated in children with ESRD. Using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) approach, we measured endothelial-specific and progenitor-associated genes VE-cadherin (VE-C), CD146, CD31, tyrosine-protein kinase receptor (Tie-2), Flk1, CD133, and growth factors promoting EPC function, vascular endothelial growth factor (VEGF), erythropoietin (EPO), and stromal cell-derived factor-1 (SDF-1) in the blood of pediatric patients undergoing hemodialysis and after transplantation. Patients' metabolic parameters were correlated with EPC marker gene levels. Compared with controls, circulating VE-cadherin, CD146, Flk1, VEGF, and EPO RNA levels were decreased in ESRD and normalized in transplanted patients. Levels of VE-cadherin, which were the most significantly reduced in ESRD (p = 0.001) inversely correlated in all of the patient population with serum urea and creatinine concentration, whereas among the ESRD group showed an inverse correlation with diastolic blood pressure (BP), interventricular septum thickness (IVST), and left ventricular mass index. Pediatric ESRD patients may have lower angiogenic potential and increased cardiovascular morbidity, because of decreased expression of circulating endothelial cell specific transcripts. Prospective studies are required to link this expression pattern and its restoration in transplanted patients to cardiovascular outcome.

The human liver is a complex tissue consisting of epithelial, endothelial, hematopoietic, and mesenchymal elements that probably derive from multiple lineage-committed progenitors, but no comprehensive study aimed at identifying and characterizing intrahepatic precursors has yet been published. Cell suspensions for this study were obtained by enzymatic digestion of liver specimens taken from 20 patients with chronic liver disease and 13 multiorgan donors. Stem and progenitor cells were first isolated, amplified, and characterized ex vivo according to previously validated methods, and then optimized flow cytometry was used to assess their relative frequencies and characterize their immunophenotypes in the clinical specimens. Stem and progenitor cells committed to hematopoietic, endothelial, epithelial, and mesenchymal lineages were clearly identifiable in livers from both healthy and diseased subjects. Within the mononuclear liver cell compartment, epithelial progenitors [epithelial cell adhesion molecule (EpCAM)(+)/CD49f(+)/CD29(+)/CD45(-)] accounted for 2.7-3.5% whereas hematopoietic (CD34(+)/CD45(+)), endothelial [vascular endothelial growth factor-2 (KDR)(+)/CD146(+)/CD45(-)], and mesenchymal [CD73(+)/CD105(+)/CD90 (Thy-1)(+)/CD45 (-)] stem cells and progenitors accounted for smaller fractions (0.02-0.6%). The patients' livers had higher percentages of hematopoietic and endothelial precursors than those of the donors. In conclusion, we identified and characterized precursors committed to four different lineages in adult human liver. We also optimized a flow cytometry approach that will be useful in exploring the contribution of these cells to the pathogenesis of liver disease.

BACKGROUND

Previous studies have shown that zinc chloride (ZnCl(2)) can induce metallthionein (MT) in the liver and kidney to protect tissues against toxicants and shows a better corneal wound healing than conventional drugs do. We hypothesized that ZnCl(2) can promote odontogenesis of dental pulp stem cells (DPSCs) via MT. The purpose of this study was to investigate the effects of ZnCl(2) on human DPSCs and the expression of MT.

METHODS

DPSCs were isolated by flow cytometry with selective surface marker CD146 and STRO-1. After they grew into confluence, DPSCs were induced into odontoblasts with or without ZnCl(2) supplemented in the culture medium for 21 days. The effect of ZnCl(2) on DPSCs differentiation was examined followed by alkaline phosphatase staining/activity and quantitative real-time polymerase chain reaction analysis.

RESULTS

By treating DPSCs with ZnCl(2), the duration of mineralization was shortened and expressions of differentiation markers into odontoblasts were more significant than those without ZnCl(2) stimulation. Besides, the MT gene expression was increased with the increasing expressions of odontoblasts' markers after treated with ZnCl(2).

CONCLUSIONS

This was the first report that ZnCl(2) could promote odontoblastic differentiation of DPSCs through the up-regulation of gene MT.

OBJECTIVE

5-fluorouracil (5FU) and its prodrug, capecitabine, can damage endothelial cells, whilst endothelial integrity is preserved by glucagon-like peptide 1 (GLP-1). Here, we studied the effect of 5FU on endothelial senescence and whether GLP-1 antagonizes it.

METHODS

EA.hy926 cells were exposed to 5FU or sera from patients taking capecitabine, with or without pre-incubation with GLP-1. Senescence was identified by expression of senescence-associated β-galactosidase and p16INK4a and reduced cell proliferation. Soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and CD146 (marker of endothelial injury) were measured by ELISA before and at completion of capecitabine chemotherapy. RT-PCR, western blotting, functional experiments with signalling inhibitors and ERK1/2 silencing were performed to characterize 5FU-induced phenotype and elucidate the pathways underlying 5FU and GLP-1 activity.

RESULTS

Both 5FU and sera from capecitabine-treated patients stimulated endothelial cell senescence. 5FU-elicited senescence occurred via activation of p38 and JNK, and was associated with decreased eNOS and SIRT-1 levels. Furthermore, 5FU up-regulated VCAM1 and TYMP (encodes enzyme activating capecitabine and 5FU), and sVCAM-1 and CD146 concentrations were higher after than before capecitabine chemotherapy. A non-significant trend for higher ICAM1 levels was also observed. GLP-1 counteracted 5FU-initiated senescence and reduced eNOS and SIRT-1 expression, this protection being mediated by GLP-1 receptor, ERK1/2 and, possibly, PKA and PI3K.

CONCLUSIONS

5FU causes endothelial cell senescence and dysfunction, which may contribute to its cardiovascular side effects. 5FU-triggered senescence was prevented by GLP-1, raising the possibility of using GLP-1 analogues and degradation inhibitors to treat 5FU and capecitabine vascular toxicity.

UNASSIGNED

This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

Chondroitin sulphate (CS) has long been used to treat osteoarthritis. Some investigations have also shown that the treatment with CS could reduce coronary events in patients with heart disease but no studies have identified the mechanistic role of these therapeutic effects. We aimed to investigate how the treatment with CS can interfere with the progress of atherosclerosis. The aortic arch, thoracic aorta and serum were obtained from apolipoprotein E (ApoE) knockout mice fed for 10 weeks with high-fat diet and then treated with CS (300 mg/kg, n = 15) or vehicle (n = 15) for 4 weeks. Atheromatous plaques were highlighted in aortas with Oil Red staining and analysed by microscopy. ApoE knockout mice treated with CS exhibited attenuated atheroma lesion size by 68% as compared with animals receiving vehicle. Serum lipids, glucose and C-reactive protein were not affected by treatment with CS. To investigate whether CS locally affects the inflamed endothelium or the formation of foam cells in plaques, human endothelial cells and monocytes were stimulated with tumour necrosis factor α or phorbol myristate acetate in the presence or absence of CS. CS reduced the expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 and ephrin-B2 and improved the migration of inflamed endothelial cells. CS inhibited foam cell formation in vivo and concomitantly CD36 and CD146 expression and oxidized low-density lipoprotein uptake and accumulation in cultured activated human monocytes and macrophages. Reported cardioprotective effects of CS may arise from modulation of pro-inflammatory activation of endothelium and monocytes and foam cell formation.

Human and mouse endometrium undergo dramatic cellular reorganization during pregnancy and postpartum. Somatic stem cells maintain homeostasis of the tissue by providing a cell reservoir for regeneration. We hypothesized that endometrial cells with quiescent properties (stem/progenitor cells) were involved in the regeneration of the endometrial tissue. Given that stem cells divide infrequently, they can retain the DNA synthesis label [bromodeoxyuridine (BrdU)] after a prolonged chase period. In this study, prepubertal mice were pulsed with BrdU and after a 6-week chase a small population of label-retaining stromal cells (LRSC) was located primarily beneath the luminal epithelium, adjacent to blood vessels, and near the endometrial-myometrial junction. Marker analyses suggested that they were of mesenchymal origin expressing CD44(+), CD90(+), CD140b(+), CD146(+), and Sca-1(+). During pregnancy, nonproliferating LRSC predominately resided at the interimplantation/placental loci of the gestational endometrium. Immediately after parturition, a significant portion of the LRSC underwent proliferation (BrdU(+)/Ki-67(+)) and expressed total and active β-catenin. The β-catenin expression in the LRSC was transiently elevated at postpartum day (PPD) 1. The proliferation of LRSC resulted in a significant decline in the proportion of LRSC in the postpartum uterus. The LRSC returned to dormancy at PPD7, and the percentage of LRSC remained stable thereafter until 11 weeks. This study demonstrated that LRSC can respond efficiently to physiological stimuli upon initiation of uterine involution and return to its quiescent state after postpartum repair.

BACKGROUND

Adiponectin may be used for assessing the risk of coronary artery disease (CAD) and may be related to the development of acute coronary syndrome. Decreased adiponectin has been associated with some risk factors for cardiovascular diseases such as male sex, obesity and diabetes mellitus. Adiponectin has antiatherogenic properties and attenuates endothelial inflammatory responses. CD146, a novel cell adhesion molecule, is localized at the endothelial junction. In kidney allograft recipients, endothelial dysfunction and atherosclerosis are almost universal. The aim of this cross-sectional study was to evaluate possible relations between adiponectin, CD146, and other markers of endothelial cell injury in 82 stable kidney transplant recipients (mean age 45 years, mean time after transplantation 47 months) with and without CAD.

METHODS

Adiponectin and markers of endothelial injury: CD146, von Willebrand factor, thrombomodulin, ICAM, CD40L, P-selectin and other hemostatic markers were assessed using commercially available kits.

RESULTS

Patients with CAD had evidence of more pronounced endothelial dysfunction, procoagulant state and lower adiponectin than patients without CAD. Adiponectin correlated significantly, in univariate analysis, with CD146 (r = 0.29, p = 0.009), thrombomodulin (r = 0.37, p = 0.001), protein Z (r = -0.25, p = 0.03), BMI (r = -0.26, p = 0.047), serum creatinine (r = 0.26, p = 0.02) and urea (r = 0.38, p = 0.001). CD146 correlated significantly with von Willebrand factor (r = 0.33, p = 0.002), thrombomodulin (r = 0.25, p = 0.025), age (r = 0.34, p = 0.001), platelets (r = -0.33, p = 0.002), serum urea (r = 0.24, p = 0.039), cholesterol (r = 0.24, p = 0.046), ICAM (r = 0.23, p = 0.036), protein C activity (r = -0.26, p = 0.019) and tended to correlate with serum creatinine and time after transplantation. In multivariate linear regression, independent predictors of adiponectin were CD146, thrombomodulin and urea, and of CD146 was mainly age of patients.

CONCLUSIONS

Endothelial dysfunction and procoagulant state are more pronounced in kidney transplant recipients with CAD, particularly in those with lower GFR. In kidney transplant recipients, markers of endothelial cell injury are significantly increased relative to healthy volunteers. Elevation of adiponectin may be a defense mechanism against endothelial damage, reflected by elevated CD146 and thrombomodulin.

The aim of this study was to investigate the levels of circulating endothelial microparticles (EMPs) in children with HSP and to determine whether there was a difference between patients with nephritis and those without nephritis. Twenty patients with HSP aged between 2.5 and 15 and 10 age-and sex-matched healthy controls were enrolled in the study. The HSP group was divided into two groups, including patients with nephritis (n = 9) and those without nephritis (n = 11). In all groups, circulating EMPs were enumerated by flow cytometry, after staining platelet-free plasma with PE-conjugated anti-CD144. At the same time, human umbilical vein endothelial cells (HUVEC) were incubated with the platelet-free plasma of patients with HSP and that of the control group. Then, circulating EMPs were counted in HUVEC supernatant incubated with the platelet-free plasma of patients and control groups, after staining the supernatant with PE-conjugated anti-CD146. Circulating EMPs were significantly higher in both the active and the remission period of the patient groups compared with the control subjects. In the patient group, there were no statistically significant differences in the level of circulating EMPs between patients with nephritis and those without nephritis. Both CD144 and 146+EMP in patients with HSP nephritis in the active period were substantially higher than in those remissions. CD144+EMP in the active period were substantially higher than in the remission period in patients without nephritis. We detected that circulating EMPs increased in patients with HSP in both active and remission periods. Although clinical and laboratory findings return to normal in the remission period, the increased circulating EMPs may show that the subclinical inflammatory process is continuous. We think that circulating EMPs could be used as a surrogate marker for subclinical inflammation in HSP.

Recently, the overexpression of CD146 and its potential as a therapeutic target in high-grade gliomas, the most lethal type of brain cancer, was uncovered. In this study, we describe the generation of (89)Zr-Df-YY146, a novel (89)Zr-labeled monoclonal antibody (mAb) for the targeting and quantification of CD146 expression in a mouse model of glioblastoma, using noninvasive immunoPET imaging. YY146, a high affinity anti-CD146 mAb, was conjugated to deferoxamine (Df) for labeling with the long-lived positron emitter (89)Zr (t1/2: 78.4 h). In vitro assays, including flow cytometry, immunofluorescence microscopy, and Western blot, were performed with two glioblastoma cell lines, U87MG and U251, to determine their CD146 expression levels. Also, YY146 and Df-YY146's CD146-binding affinities were compared using flow cytometry. In vivo CD146-targeting of (89)Zr-Df-YY146 was evaluated by sequential PET imaging, in athymic nude mice bearing subcutaneously implanted U87MG or U251 tumors. CD146 blocking, ex vivo biodistribution, and histological studies were carried out to confirm (89)Zr-Df-YY146 specificity, as well as the accuracy of PET data. In vitro studies exposed elevated CD146 expression levels in U87MG cells, but negligible levels in U251 cells. Flow cytometry revealed no differences in affinity between YY146 and Df-YY146. (89)Zr labeling of Df-YY146 proceeded with excellent yield (∼80%), radiochemical purity (>95%), and specific activity (∼44 GBq/μmol). Longitudinal PET revealed prominent and persistent (89)Zr-Df-YY146 uptake in mice bearing U87MG tumors that peaked at 14.00 ± 3.28%ID/g (n = 4), 48 h post injection of the tracer. Conversely, uptake was significantly lower in CD146-negative U251 tumors (5.15 ± 0.99%ID/g, at 48 h p.i.; n = 4; P < 0.05). Uptake in U87MG tumors was effectively blocked in a competitive inhibition experiment, corroborating the CD146 specificity of (89)Zr-Df-YY146. Finally, ex vivo biodistribution validated the accuracy of PET data and histological examination successfully correlated tracer uptake with in situ CD146 expression. Prominent, persistent, and specific uptake of (89)Zr-Df-YY146 was observed in brain tumors, demonstrating the potential of this radiotracer for noninvasive PET imaging of CD146 expression. In a future clinical scenario, (89)Zr-Df-YY146 may serve as a tool to guide intervention and assess response to CD146-targeted therapies.

The association between breast cancer initiation and prolonged exposure to estrogen suggests that this hormone may also have an etiologic role in such a process. On the other hand, many studies have found an association between human cancer and exposure to agricultural pesticides such as parathion, an organophosphorous pesticide used in agriculture to control mosquito plagues. However, the key factors behind the initiation of breast cancer remain to be elucidated. The aim of this study was to determine the effect of 17beta estradiol (estrogen) and parathion on protein expression in cell transformation of human breast epithelial cells in vitro. Estrogen and parathion alone and in combination induced malignant transformation of an immortalized human breast epithelial cell line, MCF-I0F, as indicated by anchorage independency and invasive capabilities. The results indicate that a combination of estrogen and parathion increased the expression of related cell adhesion proteins such as Dvl, Notch, CD146 and beta catenin. In conclusion, it can be suggested that pesticides affect human breast cell adhesion changes indicative of transformation.

Long-term self-renewing hematopoietic stem cell (LT-HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT-HSC and a number of niche-associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin+ Lepr + Sca-1+ CD146+ and could be stably propagated with high colony-forming unit fibroblast re-cloning efficiency. MSC synergized with SCF and IL-11 to support a 20-fold expansion in true LT-HSC after 10-days of in vitro coculture. Optimal stimulation of LT-HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self-renewal-promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT-HSC homeostasis in vivo. Stem Cells 2017;35:473-484.

Cell adhesion molecules (CAMs) are crucial effectors for the development and maintenance of the nervous system. Mutations in human CAM genes are linked to brain disorders and psychological diseases, and CAM knockout mice always exhibit similar behavioral abnormalities. CD146 is a CAM of the immunoglobulin superfamily that interacts with Neurite Outgrowth Factor and involved in neurite extension in vitro. However, little is known about its in vivo function in the nervous system. In this study, we used a murine CD146 nervous system knockout (CD146(ns-ko)) model. We found that the brains of some CD146(ns-ko) mice were malformed with small olfactory bulbs. CD146(ns-ko) mice exhibited lower body weights and smaller food intake when compared with wild type littermates. Importantly, behavior tests revealed that CD146(ns-ko) mice exhibited significant decreased locomotor activity and impaired capacity for spatial learning and memory. Our results demonstrate that CD146 is important for mammalian nervous system development and proper behavior patterns.

Netrin-1, a classic neuronal guidance cue, can promote angiogenesis under certain developmental and pathological conditions, but key receptors on vascular endothelium have remained elusive. A recent study published in Cell Research by Tu et al. reveals that CD146, an endothelial receptor of the immunoglobulin superfamily, binds netrin-1 with high affinity and may play an important role in regulating angiogenesis.

Patients with Alzheimer's disease (AD) have blood-brain barrier (BBB) dysfunction. Methods to study cells of the BBB in vivo would facilitate analyses of neurovascular damage in early AD. Thus, we conducted a pilot study to investigate if brain-derived endothelial cells (BDCECs) could be identified from a cell population of circulating endothelial cells (CECs). Peripheral blood was sampled from early AD patients (n = 9), patients with vascular diseases (myocardial infarction (n = 8) and ischemic stroke (n = 8)), and healthy controls (n = 8). We enumerated CD34+/CD146+/CD45- cells (CECs) and Glucose transporter-1 (Glut1+ CECs (BDCECs)) by flow cytometry. We found that BDCECs formed a separate, aggregate cell population. Glut1 expression on BDCECs, measured by the median fluorescence intensity, was significantly decreased in patients with AD compared to both the healthy controls and patients with myocardial infarction ((p < .05, Kruskal-Wallis, Dunn's post hoc test). We found no significant differences in cell numbers. Our study shows that isolation of BDCECs offers a promising non-invasive tool to investigate cells derived from the BBB. Downregulation of Glut1 at the mild stages of AD suggests that agents that increase Glut1 levels may be therapeutic candidates to improve energy availability to the brain.

OBJECTIVE

The structural and functional integrity of bone-periodontal ligament (PDL)-cementum complex stems from the load-bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for the maintenance of a bone-PDL-cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses.

METHODS

Multilabeling technique was performed on hemimandibles of 3 wk and 3 mo-old scleraxis-GFP transgenic mice for CD146, CD31, NG2, osterix and bone sialoprotein. Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts and PDL cell cytoskeleton.

RESULTS

CD146+ cells adjacent to CD31+ vasculature were identified at PDL-bone enthesis. NG2+ cells were located at coronal bone-PDL and apical cementum-PDL entheses in the 3-wk-old group, but at 3 mo, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and osterix were colocalized at the osteoid and cementoid regions of the PDL-bone and PDL-cementum entheses. Bone sialoprotein was prominent at the apical region of 3-wk-old mice. The directionality of collagen fibers, fibroblasts and their cytoskeleton overlapped, except in the apical region of 3 wk.

CONCLUSIONS

Colocalization of biomolecules at zones of the PDL adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load-bearing bone-PDL-tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from a vascular origin toward osteo- and cemento-blastic lineages at the PDL-bone and PDL-cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex.

Endometrial CD146+ cells were purified, using magnetic activated cell sorting, and then embedded and cultured in a collagen-matrigel scaffold on top of myometrial smooth muscle cells for 10 days. At the end of culture period, the differentiation and formation of the epithelial-like cells were confirmed by morphological and ultrastructural evaluations, and analysis by reverse transcription polymerase chain reaction of the specific expression of genes: osteopontin (SPP1), matrix metalloproteinase 2, zonula occludens 1, laminin alpha 2 and collagen type IV; and by western blotting of CD9 protein. The results showed that the human endometrial mesenchymal CD146+ cells were able to produce endometrial glandular tube-like structures in vitro. Ultrastructural observation revealed some projections on the apical surfaces, appearance of basal lamina-like structures on the basal surface, and tight junctions and desmosomes on the lateral surfaces of the epithelial-like cells. The expression of studied genes at RNA level and CD9 at protein level confirmed the formation of endometrial epithelial-like cells. This culture system may have potential applications in cell therapy and in studies on human embryo implantation.

Human urine-derived stem cells (hUSCs) are a potential stem cell source for cell therapy. However, the effect of hUSCs on glucose metabolism regulation in type 1 diabetes was not clear. Therefore, the aim of the study was to evaluate whether hUSCs have protective effect on streptozotocin (STZ)-induced diabetes. hUSCs were extracted and cultivated with a special culture medium. Flow cytometry analysis was applied to detect cell surface markers. BALB/c male nude mice were either injected with high-dose STZ (HD-STZ) or multiple low-dose STZ (MLD-STZ). Serum and pancreatic insulin were measured, islet morphology and its vascularization were investigated. hUSCs highly expressed CD29, CD73, CD90 and CD146, and could differentiate into, at least, bone and fat in vitro. Transplantation of hUSCs into HD-STZ treated mice prolonged the median survival time and improved their blood glucose, and into those with MLD-STZ improved the glucose tolerance, islet morphology and increased the serum and pancreas insulin content. Furthermore, CD31 expression increased significantly in islets of BALB/c nude mice treated with hUSCs compared to those of un-transplanted MLD-STZ mice. hUSCs could improve the median survival time and glucose homeostasis in STZ-treated mice through promoting islet vascular regeneration and pancreatic beta-cell survival.

Purpose/Aim: Cementogenesis is a critical step in periodontal tissue regeneration given the essential role of cementum in anchoring teeth to the alveolar bone. This study is designed to achieve integrated cementum formation on the root surfaces of human teeth using growth factor-releasing scaffolds with periodontal ligament stem/progenitor cells (PDLSCs).

Human PDLSCs were sorted by CD146 expression, and characterized using CFU-F assay and induced multi-lineage differentiation. Polycaprolactone scaffolds were fabricated using 3D printing, embedded with poly(lactic-co-glycolic acids) (PLGA) microspheres encapsulating connective tissue growth factor (CTGF), bone morphogenetic protein-2 (BMP-2), or bone morphogenetic protein-7 (BMP-7). After removing cementum on human tooth roots, PDLSC-seeded scaffolds were placed on the exposed dentin surface. After 6-week culture with cementogenic/osteogenic medium, cementum formation and integration were evaluated by histomorphometric analysis, immunofluorescence, and qRT-PCR.

Periodontal ligament (PDL) cells sorted by CD146 and single-cell clones show a superior clonogenecity and multipotency as compared with heterogeneous populations. After 6 weeks, all the growth factor-delivered groups showed resurfacing of dentin with a newly formed cementum-like layer as compared with control. BMP-2 and BMP-7 showed de novo formation of tissue layers significantly thicker than all the other groups, whereas CTGF and BMP-7 resulted in significantly improved integration on the dentin surface. The de novo mineralized tissue layer seen in BMP-7-treated samples expressed cementum matrix protein 1 (CEMP1). Consistently, BMP-7 showed a significant increase in CEMP1 mRNA expression.

Our findings represent important progress in stem cell-based cementum regeneration as an essential part of periodontium regeneration.