Multiple sclerosis is a chronic auto-inflammatory disease of the central nervous system affecting patients worldwide. Neuroinflammation in multiple sclerosis is mainly driven by peripheral immune cells which invade the central nervous system and cause neurodegenerative inflammation. To enter the target tissue, immune cells have to overcome the endothelium and transmigrate into the tissue. Numerous molecules mediate this process and, as they determine the tissue invasiveness of immune cells, display great therapeutic potential. Melanoma cell adhesion molecule (MCAM) is a membrane-anchored glycoprotein expressed by a subset of T-cells and MCAM+ T-cells have been shown to contribute to neuroinflammation in multiple sclerosis. The role of the MCAM molecule for brain invasion, however, remained largely unknown. In order to investigate the role of the MCAM molecule on T-cells, we used different in vitro and in vivo assays, including ex vivo flow chambers, biochemistry and microscopy experiments of the mouse brain. We demonstrate that MCAM directly mediates adhesion and that the engagement of MCAM induces intracellular signaling leading to β1-integrin activation on human T-cells. Furthermore, we show that MCAM engagement triggers the phosphorylation of PLCγ1 which is required for integrin activation and thus amplification of the cellular adhesive potential. To confirm the physiological relevance of our findings in vivo, we demonstrate that MCAM plays an important role in T-cell recruitment into the mouse brain. In conclusion, our data demonstrate that MCAM expressed on T-cells acts as an adhesion molecule and a signaling receptor that may trigger β1-integrin activation via PLCγ1 upon engagement.

Keywords: integrin; PLCγ1 activation; T-cell; adhesion; melanoma cell adhesion molecule/CD146; multiple sclerosis; neuroinflammation; recruitment.

Metastatic uveal melanoma (mUM) to the liver is incurable. Transcriptome profiling of 40 formalin-fixed paraffin-embedded mUM liver resections and 6 control liver specimens was undertaken. mUMs were assessed for morphology, nuclear BAP1 (nBAP1) expression, and their tumour microenvironments (TME) using an "immunoscore" (absent/altered/high) for tumour-infiltrating lymphocytes (TILs) and macrophages (TAMs). Transcriptomes were compared between mUM and control liver; intersegmental and intratumoural analyses were also undertaken. Most mUM were epithelioid cell-type (75%), amelanotic (55%), and nBAP1-ve (70%). They had intermediate (68%) or absent (15%) immunoscores for TILs and intermediate (53%) or high (45%) immunoscores for TAMs. M2-TAMs were dominant in the mUM-TME, with upregulated expression of ANXA1, CD74, CXCR4, MIF, STAT3, PLA2G6, and TGFB1. Compared to control liver, mUM showed significant (p < 0.01) upregulation of 10 genes: DUSP4, PRAME, CD44, IRF4/MUM1, BCL2, CD146/MCAM/MUC18, IGF1R, PNMA1, MFGE8/lactadherin, and LGALS3/Galectin-3. Protein expression of DUSP4, CD44, IRF4, BCL-2, CD146, and IGF1R was validated in all mUMs, whereas protein expression of PRAME was validated in 10% cases; LGALS3 stained TAMs, and MFGEF8 highlighted bile ducts only. Intersegmental mUMs show differing transcriptomes, whereas those within a single mUM were similar. Our results show that M2-TAMs dominate mUM-TME with upregulation of genes contributing to immunosuppression. mUM significantly overexpress genes with targetable signalling pathways, and yet these may differ between intersegmental lesions.

Keywords: DUSP4; MUM1; immunotherapy; metastatic uveal melanoma; transcriptome profiling.

There are various types of adipose tissue in the human body, and their morphology is known to be closely related to cell function and metabolism. However, the functional differences among the mesenchymal stromal cells (MSCs) of different abdominal adipose tissues have not been clearly elucidated.

MSCs were isolated from different abdominal adipose tissues according to their regional distribution and included superficial subcutaneous, deep subcutaneous, omentum, mesentery and retroperitoneal MSCs. The immunophenotype, proliferative ability and angiogenic function of these MSCs were compared based on flow cytometry analysis, CCK-8 proliferation, in vitro differentiation, tubule formation and in vivo plug assay.

The plastic adherence, cell morphology and general immunophenotype are similar among the MSCs. However, subcutaneous adipose tissue-derived MSCs have a faster growth rate and a higher level of CD146 expression than the other MSCs. Moreover, according to the fluorescence-activated cell sorting (FACS) enrichment procedure, the expression level of CD146 is positively related to the growth rate and angiogenic capability of MSCs.

MSCs in adipose tissue showed slightly different characteristics depending on their location of origin, and they possessed different angiogenic abilities that were mediated by the expression of CD146. This study provides evidence that subcutaneous adipose tissue is the most appropriate source of MSCs for therapeutic cell transplantation in vascular disease.

Lipedema is a hormone-related disease of women characterized by enlargement of the extremities caused by subcutaneous deposition of adipose tissue. In healthy patients application of autologous adipose tissue-derived cells has shown great potential in several clinical studies for engrafting of soft tissue reconstruction in recent decades. The majority of these studies have used the stromal vascular fraction (SVF), a heterogeneous cell population containing adipose-derived stromal/stem cells (ASC), among others. Because cell identity and regenerative properties might be affected by the health condition of patients, we characterized the SVF cells of 30 lipedema patients in comparison to 22 healthy patients.

SVF cells were analyzed regarding cell yield, viability, adenosine triphosphate content, colony forming units and proliferative capacity, as well as surface marker profile and differentiation potential in vitro.

Our results demonstrated a significantly enhanced SVF cell yield isolated from lipedema compared with healthy patients. In contrast, the adipogenic differentiation potential of SVF cells isolated from lipedema patients was significantly reduced compared with healthy patients. Interestingly, expression of the mesenchymal marker CD90 and the endothelial/pericytic marker CD146 was significantly enhanced when isolated from lipedema patients.

The enhanced number of CD90+ and CD146+ cells could explain the increased cell yield because the other tested surface marker were not reduced in lipedema patients. Because the cellular mechanism and composition in lipedema is largely unknown, our findings might contribute to a better understanding of its etiology.

Multipotent/Mesenchymal stromal cells (MSC) exist within a variety of postnatal tissues, however global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSC as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSC (Panc-MSC) with a classical phenotype (CD90+/CD73+/CD105+/CD45-/CD31-). Both MSC subtypes expressed mesenchymal markers Vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSC. Accordingly, these Vimentin^high /Nestin^high cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSC and BM-MSC, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSC. Panc-MSC also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSC showed restricted responses to adipogenic stimuli in vitro, although both MSC-types demonstrated trilineage differentiation. In contrast, Panc-MSC demonstrated accelerated growth kinetics and competency to pro-neurogenic stimuli in vitro. The secretome of Panc-MSC was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSC, Panc-MSC conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC-types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSC as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease. © AlphaMed Press 2019 SIGNIFICANCE STATEMENT: Multipotent/mesenchymal stromal cells (MSC) have been subcultured from various tissue sources, leading to a spectrum of properties reflective of tissue microenvironment or developmental origin. As a result, MSC possess a diverse range of therapeutic benefits, including immunomodulatory, angiogenic and tissue regenerative properties. Herein, we utilize label-free mass spectrometry and functional analyses to characterize the proteome and secretome of MSC populations established from human pancreas tissue (Panc-MSC). Specifically, Panc-MSC demonstrated a unique Vimentin^high /Nestin^high proteome restricted from adipogenesis, yet secreted proteomic effectors associated with endothelial cell chemotaxis, angiogenesis and islet regeneration. These foundational analyses begin to elucidate secretory functions of Panc-MSC as a possible therapeutic agent for regenerative medicine applications.

Human umbilical cord blood (CB) has attracted much attention as a reservoir for functional hematopoietic stem and progenitor cells, and, recently, as a source of blood-borne fibroblasts (CB-BFs). Previously, we demonstrated that bone marrow stromal cell (BMSC) and CB-BF pellet cultures make cartilage in vitro Furthermore, upon in vivo transplantation, BMSC pellets remodelled into miniature bone/marrow organoids. Using this in vivo model, we asked whether CB-BF populations that express characteristics of the hematopoietic stem cell (HSC) niche contain precursors that reform the niche. CB ossicles were regularly observed upon transplantation. Compared with BM ossicles, CB ossicles showed a predominance of red marrow over yellow marrow, as demonstrated by histomorphological analyses and the number of hematopoietic cells isolated within ossicles. Marrow cavities from CB and BM ossicles included donor-derived CD146-expressing osteoprogenitors and host-derived mature hematopoietic cells, clonogenic lineage-committed progenitors and HSCs. Furthermore, human CD34+ cells transplanted into ossicle-bearing mice engrafted and maintained human HSCs in the niche. Our data indicate that CB-BFs are able to recapitulate the conditions by which the bone marrow microenvironment is formed and establish complete HSC niches, which are functionally supportive of hematopoietic tissue.

Endothelial colony-forming cells (ECFC) are currently considered as a promising cell population for the pre-endothelialization or pre-vascularization of tissue-engineered constructs, including small-diameter biodegradable vascular grafts. However, the extent of heterogeneity between ECFC and mature vascular endothelial cells (EC) is unclear. Here, we performed a transcriptome-wide study to compare gene expression profiles of ECFC, human coronary artery endothelial cells (HCAEC), and human umbilical vein endothelial cells (HUVEC). Characterization of the abovementioned cell populations was carried out by immunophenotyping, tube formation assay, and evaluation of proliferation capability while global gene expression profiling was conducted by means of RNA-seq. ECFC were similar to HUVEC in terms of immunophenotype (CD31⁺vWF⁺KDR⁺CD146⁺CD34^-CD133^-CD45^-CD90^-) and tube formation activity yet had expectedly higher proliferative potential. HCAEC and HUVEC were generally similar to ECFC with regards to their global gene expression profile; nevertheless, ECFC overexpressed specific markers of all endothelial lineages (NRP2, NOTCH4, LYVE1), in particular lymphatic EC (LYVE1), and had upregulated extracellular matrix and basement membrane genes (COL1A1, COL1A2, COL4A1, COL4A2). Proteomic profiling for endothelial lineage markers and angiogenic molecules generally confirmed RNA-seq results, indicating ECFC as an intermediate population between HCAEC and HUVEC. Therefore, gene expression profile and behavior of ECFC suggest their potential to be applied for a pre-endothelialization of bioartificial vascular grafts, whereas in terms of endothelial hierarchy they differ from HCAEC and HUVEC, having a transitional phenotype.

BACKGROUND

Endothelial progenitor cells (EPC) are markers of endothelial injury and may serve as a surrogate marker for vascular repair in interventional clinical trials. Objectives of this study were to modify a method of isolation of peripheral blood mononuclear cells (PBMC) and enumeration of EPC and mature endothelial cells (EC) from peripheral blood and to evaluate influence of cryopreservation on viability of PBMC and on numbers of EPC and EC.

METHODS

EPC and EC were analyzed in healthy volunteers in freshly isolated PBMC collected in CPT (cell preparation tubes) and in PBMC cryopreserved with: 1) Gibco Recovery™ Cell Culture Freezing Medium, 2) custom freezing medium. Viability of PBMC was tested using DAPI. EPC were gated for CD45- CD34+CD133+/-VEGFR2+/- and EC were gated for CD45-CD146+CD34+/-VEGFR2+/-.

RESULTS

Cryopreservation for 7 days at -80°C decreased viable PBMC from 94 ± 0.5% (fresh) to 84 ± 4% (the custom medium) and to 69 ± 8% (Gibco medium), while cryopreservation at -65°C decreased viability to 60 ± 6% (p<0.001, the custom medium) and 49 ± 5% (p<0.001, Gibco medium). In fresh samples early EPC (CD45- CD34+CD133+VEGFR2+) were enumerated as 0.2 ± 0.06%, late EPC(CD45-CD146+CD34+VEGFR2+) as 0.6 ± 0.1% and mature EC (CD45-CD146+CD34-VEGFR2+) as 0.8 ± 0.3%of live PBMC. Cryopreservation with Gibco and the custom freezing medium at -80°C for 7 days decreased numbers EPC and EC, however, this decrease was not statistically significant.

CONCLUSIONS

Our data indicate that cryopreservation at -80°C for 7 days decreases, although not significantly, viability of PBMC and numbers of subsets of EC and EPC. This method may provide an optimized approach to isolation and short-term cryopreservation of subsets of EPC and of mature EC suitable for multicenter trials.

Cerebral malaria (CM) is a life-threatening diffuse encephalopathy caused by Plasmodium falciparum, in which the destruction of the blood-brain barrier (BBB) is the main cause of death. However, increasing evidence has shown that antimalarial drugs, the current treatment for CM, do little to protect against CM-induced BBB damage. Therefore, a means to alleviate BBB dysfunction would be a promising adjuvant therapy for CM. The adhesion molecule CD146 has been reported to be expressed in both endothelial cells and proinflammatory immune cells and mediates neuroinflammation. Here, we demonstrate that CD146 expressed on BBB endothelial cells but not immune cells is a novel therapeutic target in a mouse model of experimental cerebral malaria (eCM). Endothelial CD146 is upregulated during eCM development and facilitates the sequestration of infected red blood cells (RBCs) and/or proinflammatory lymphocytes in CNS blood vessels, thereby promoting the disruption of BBB integrity. Mechanistic studies showed that the interaction of CD146 and Galectin-9 contributes to the aggregation of infected RBCs and lymphocytes. Deletion of endothelial CD146 or treatment with the anti-CD146 antibody AA98 prevents severe signs of eCM, such as limb paralysis, brain vascular leakage, and death. In addition, AA98 combined with the antiparasitic drug artemether improved the cognition and memory of mice with eCM. Taken together, our findings suggest that endothelial CD146 is a novel and promising target in combination with antiparasitic drugs for future CM therapies.

Keywords: BBB; CD146; experimental cerebral malaria.

Human trophoblasts consist of two main cell lineages, villous trophoblasts (VT) and extravillous trophoblasts (EVT). To identify the molecules which are involved in EVT differentiation, we have raised a monoclonal antibody (mAb) designated CHL1, by immunizing a mouse against human chorion laeve which is composed of EVT. By immunohistochemical analysis, the CHL1 antigen was found to be expressed on the majority of EVT but not on VT in addition to its expression on endothelial and myometrial cells. A subsequent cDNA panning method revealed that the CHL1 antigen was identical to melanoma cell adhesion molecule (MCAM, Mel-CAM, S-endo 1 or MUC18/CD146), which has been previously reported as one of the EVT markers. MCAM expression on JEG3 cells, a human choriocarcinoma-derived cell line, was significantly enhanced when they were co-cultured with isolated human decidual tissue. Various cytokines and growth factors that were reportedly present in decidual tissue failed to increase MCAM expression in JEG3 cells, but decidua-induced MCAM expression in JEG3 cells was attenuated by the addition of protein kinase A inhibitor H89. In addition, cAMP, which is known to stimulate differentiation of VT, enhanced MCAM expression in JEG3 cells. Its promoting effect on MCAM expression was also observed in human chorionic villous explant cultures. These findings suggest that a cAMP-dependent intracytoplasmic signalling pathway is involved in the differentiation mechanism of human EVT.

Human decidual stromal cells (DSCs) play a key role in maternal-fetal interactions. Precursors of DSCs (preDSCs) localize around vessels in both the endometrium and decidua. Previous studies suggested a relationship between preDSCs and pericytes because these cells share a perivascular location, alpha smooth muscle actin (α-SM actin) expression and the ability to contract under the effects of cytokines.

To further study this relationship, we established 15 human preDSC lines and 3 preDSC clones. The preDSC lines and clones were tested by flow cytometry with a panel of 29 monoclonal antibodies, 14 of which are pericyte markers. The expression of angiogenic factors was determined by RT-PCR, chemotactic activity was studied with the migration assay, and cell contractility was evaluated with the collagen cell contraction assay. Confocal microscopy was used to study decidual sections.

Under the effect of progesterone and cAMP, these lines decidualized in vitro: the cells became rounder and secreted prolactin, a marker of physiological DSC differentiation (decidualization). The antigen phenotype of these preDSC lines and clones was fully compatible with that reported for pericytes. PreDSC lines displayed pericyte characteristics: they expressed angiogenic factors and showed chemotactic and cytokine-induced contractile activity. Confocal microscopic examination of decidual sections revealed the expression of antigens detected in preDSC lines: α-SM actin colocalized with CD146, CD140b, MFG-E8, nestin, and STRO-1 (all of which are pericyte markers) in cells located around the vessels, a distinctive location of preDSCs and pericytes.

Taken together, our results show that preDSCs are pericyte-like cells.

The significance of indoleamine 2,3-dioxygenase-1 (IDO1) has been studied in various types of tumors, but the relationship between IDO1 and tumor angiogenesis needs further delineation. We aimed to clarify the relationship between tumor angiogenesis and IDO1 expression, and to explore the possibility of IDO1-targeting molecular therapy for lung cancer. For the first time, we found that silencing the IDO1 gene using small interfering RNA (siRNA) inhibits in vitro cancer cell invasion and migration. We further demonstrated that knockdown of IDO1 decreased the formation of vasculogenic mimicry. In addition to these in vitro findings, we also demonstrated that in vivo IDO1 gene silencing using short hairpin RNA (shRNA) delayed tumor onset and inhibited tumor growth in the mouse model. Immunostaining showed that IDO1 gene silencing inhibited tumor angiogenesis. Moreover, the expression of IDO1 was associated with microvessel density (MVD) labeled by CD34 and CD146. These findings indicate that IDO1 has the potential to participate in or contribute to the formation of new capillaries, supporting the applicability of IDO1-targeting molecular therapy in lung cancer.

Objectives: Various factors could interfere the biological performance of DPSCs during post-thawed process. Yet, little has been known about optimization of the recovery medium for DPSCs. Thus, our study aimed to explore the effects of adding recombinant bFGF on DPSCs after 3-month cryopreservation as well as the underlying mechanisms.

Materials and methods: DPSCs were extracted from impacted third molars and purified by MACS. The properties of CD146⁺ DPSCs (P3) were identified by CCK-8 and flow cytometry. After cryopreservation for 3 months, recovered DPSCs (P4) were immediately supplied with a series of bFGF and analysed cellular proliferation by CCK-8. Then, the optimal dosage of bFGF was determined to further identify apoptosis and TRPC1 channel through Western blot. The succeeding passage (P5) from bFGF pre-treated DPSCs was cultivated in bFGF-free culture medium, cellular proliferation and stemness were verified, and pluripotency was analysed by neurogenic, osteogenic and adipogenic differentiation.

Results: It is found that adding 20 ng/mL bFGF in culture medium could significantly promote the proliferation of freshly thawed DPSCs (P4) through suppressing apoptosis, activating ERK pathway and up-regulating TRPC1. Such proliferative superiority could be inherited to the succeeding passage (P5) from bFGF pre-stimulated DPSCs, meanwhile, stemness and pluripotency have not been compromised.

Conclusions: This study illustrated a safe and feasible cell culture technique to rapidly amplify post-thawed DPSCs with robust regenerative potency, which brightening the future of stem cells banking and tissue engineering.

Keywords: basic fibroblast growth factor; cell culture technique; cryopreservation; dental pulp stem cells; extracellular signal-regulated kinase pathway; transient receptor potential canonical 1 channel.

Human osteogenic progenitors are not precisely defined, being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells (MSCs). Notably, select human pericytes can develop into bone-forming osteoblasts. Here, we sought to define the differentiation potential of CD146⁺ human pericytes from skeletal and soft tissue sources, with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte. CD146⁺CD31^-CD45^- pericytes were derived by fluorescence-activated cell sorting from human periosteum, adipose, or dermal tissue. Periosteal CD146⁺CD31^-CD45^- cells retained canonical features of pericytes/MSC. Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo, while soft tissue pericytes did not readily. Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts, and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes. Conversely, enrichment of CXCR4⁺ pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell. In sum, human skeletal and soft tissue pericytes differ in their basal abilities to form bone. Diversity exists in soft tissue pericytes, however, and CXCR4⁺ pericytes represent an osteoblastogenic, non-adipocytic cell precursor. Indeed, enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering.

Keywords: Bone; Diseases.

OBJECTIVE

To investigate the expression of surface antigen of human periodontal ligament cells (PDLCs) and dental pulp cells (DPCs) and the impact of ex vivo expansion to the expression of surface antigen. To provide basis of proper surface antigen for further selection of homogenous stem cell subpopulation from PDLCs and DPCs.

METHODS

PDLCs and DPCs were isolated and cultured by collagenase type I and dispase. The expression of surface antigen was analyzed by immunohistochemistry and flow cytometry.

RESULTS

Positive expression of STRO-1 and CD146 were observed in PDLCs and DPCs by immunocytochemistry. Similar to DPCs, PDLCs expressed mesenchymal stem cell markers STRO-1, CD146, CD29, CD44 and CD106, and displayed negative expression for CD34 at passage 1 by flow cytometry. There were no significant difference of STRO-1, CD29 and CD44 expression level between PDLCs and DPCs (P>> 0.05). PDLCs expressed significantly higher level of CD106 and significantly lower level of CD146 than DPCs (P < 0.001). The proportion of STRO-1 and CD146 positive cells decreased steadily with passages in PDLCs and DPCs.

CONCLUSIONS

PDLCs have some similar surface antigen as DPCs, and the stem cells properties of PDLCs and DPCs decreased steadily with passages.

DHEA (dehydroepiandrosterone) is a weak androgen with proposed efficacy in the treatment of mild to moderate lupus, and possible beneficial effects on cardiovascular risk and bone mineral density. We hypothesized that treatment with 200 mg a day of Prasterone (DHEA) would improve pre-clinical measures of atherosclerosis: flow-mediated dilatation (FMD), nitroglycerin-mediated dilatation (NMD), and circulating apoptotic endothelial cells (CD 146(AnnV +)), as well markers of bone metabolism. Thirteen premenopausal female patients with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) <or=8 were enrolled in a double-blind placebo-controlled crossover trial for 22 weeks with a 6-week washout between treatment periods. Results reveal high-density lipoprotein (HDL) levels significantly decreased with Prasterone (48.5 versus 56.3 with placebo, p <or= 0.001), and there was a trend towards impairment of endothelial function with Prasterone (brachial artery FMD 3.4% versus 4.4% with placebo, mean difference -1.07, NMD 19.5% versus 24.4% with placebo, mean difference -4.9, p = NS). There were no differences between groups in SLEDAI, <em>CD146</em>( AnnV+) cells, or receptor activator for nuclear factor kB ligand (RANKL)/osteoprotegerin, although RANKL was higher after treatment with Prasterone (mean difference -29.5 units; p = 0.097). This pilot study does not support the use of Prasterone in mild lupus for prevention of atherosclerosis or osteoporosis, and confirms other findings of potentially harmful effects on lipids.

The aims of this study were to determine the presence and distribution of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR2) in dentigerous cysts compared with normal dental follicles as a control tissue and to evaluate endothelial cells and proliferating cells as indicators of angiogenic activity in these tissues.Twenty specimens histologically diagnosed as dentigerous cysts and 20 dental follicle specimens were included. Immunohistochemistry (IHC) using anti-VEGF and anti-VEGFR2 antibodies stained for the growth factor and its receptor, while anti-CD34 and anti-CD146 antibodies were used to identify endothelial cells. Anti-proliferating cell nuclear antigen (PCNA) antibody detected proliferating cells within the specimens. Slides were examined microscopically and results evaluated using kappa statistics, negative binomial regression and ordinal logistic regression.The mean age for patients with dentigerous cysts was 23 years and they were more common in males. Proteins for VEGF, VEGFR2, PCNA, CD34, and CD146 were expressed in all dentigerous cysts and dental follicles. VEGF and VEGFR2 were expressed on several cell types within the tissues, however there was a significantly greater percentage of positive staining in dentigerous cysts compared with dental follicles (odds ratio = 31.24, p < 0.001). CD34(+), CD146(+), and PCNA(+) cells were observed in both dentigerous cysts and dental follicles but for all markers there were significantly more positive cells in dentigerous cysts (p < 0.001); this was especially evident in cases associated with inflammation. PCNA was seen in most endothelial cells lining small thin walled blood vessels suggesting endothelial proliferation. There was a high level of intra- and inter-examiner agreement (kappa 0.77 and 0.75, respectively).VEGF and VEGFR2 and angiogenic activity are present in dental follicles and dentigerous cysts and may contribute to local bone resorption for tooth eruption or the development and progression of dentigerous cysts.

Highly sensitive and enzyme-free detection of melanoma adhesion molecule antigen (CD146) remains a challenge in clinical diagnosis. The prepared immunosensor, based on amination graphene (GS-NH2) and mesoporous nano-Co3O4 sheet combined with gold nanoparticles (Au/Co3O4), exhibited significantly increased electron transfer, high sensitivity and stability to CD146. Au/Co3O4 can increase the contact surface between the antibody and Au nanoparticles attached on Co3O4 than mesoporous Co3O4 only. And the mesoporous Co3O4 nanosheet can capture more biomolecules to enhance the sensitivity due to the large effective specific area. Amperometric i-t curve and electrochemical impedance spectroscopy (EIS) were used to characterize the recognition of CD146. This novel immunosensor, works well over a broad linear range of 0.01-15ng/mL, with a low detection limit of 3.4pg/mL (S/N=3). The immunosensor was evaluated for the determination of human serum sample, and received a satisfactory result. The developed immunosensor provides a promising approach for clinical research and diagnostic applications.

The cell adhesion molecule CD146 is normally located at the endothelial cell-to-cell junction and colocalizes with actin cytoskeleton. The soluble form of CD146 (sCD146) has been identified in the endothelial cell supernatant and in normal human plasma, and is increased in pathologic conditions with altered endothelial function. Soluble CD146 binding to monocytes promotes their transendothelial migration, which represents a central step in the development of atherosclerotic plaque. Since peripheral blood monocytes are characterized by a phenotypic and functional heterogeneity, with different transendothelial migration capacity, we hypothesized that monocyte subsets differently bind sCD146. Based on surface CD14 and CD16 expression monocytes were distinguished by flow cytometry (FACS) into three subsets: CD14++/CD16-, CD14++/CD16+ and CD14+/CD16+. CD16+ monocytes have been found to possess higher transendothelial migration ability. FACS analysis on blood monocytes from 30 healthy subjects revealed that higher percentages of CD14++/CD16+ (median, first and third quartile: 2.26, 1.62-3.87) and of CD14+/CD16+ (2.59, 1.28-4.80) were positive for CD146 (both p < 0.01), in comparison to CD14++/CD16- (0.66, 0.47-1.01). Moreover, in vitro treatment of ficoll separated monocytes with recombinant CD146 showed that both CD16+ subsets increased their percentage of CD146-positive events compared to CD16- monocytes (p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples of subjects from our study group and showed a correlation with percentage of CD146-positive CD14+/CD16+ monocyte subset. In this work we have demonstrated that monocyte subsets behave differently with regard to their sCD146 binding activity; because binding of CD146 influences transendothelial migration of monocytes, modulation of monocyte-CD146 interaction may represent a potential target to limit atherosclerotic plaque development.

OBJECTIVE

While most human adipose tissues, such as those located in the abdomen, hip and thigh, are of mesodermal origin, adipose tissues located in the face are of ectodermal origin. The present study has compared stem cell-related features of abdomen-derived adult stem cells (A-ASCs) with those of eyelid-derived adult stem cells (E-ASCs).

METHODS

Adipose tissue-derived cells were maintained in DMEM supplemented with 10% FBS. Before passage 6, cells were analysed using FACS, immunocytochemistry and quantitative real time PCR (qRT-PCR). To examine multi-differentiational potential, early passage ASCs were cultivated in each of a commercial Stempro(®) Differentiation kit.

RESULTS

Unlike fibroblast-like morphology of A-ASCs, E-ASCs had bipolar morphology. Both types of cell exhibited similar surface antigens, and neuronal cell-related genes and proteins. However, there were differences in mRNA expression levels of CD90 and CD146; neuron-specific enolase (NSE) and nuclear receptor-related protein 1 (Nurr1) were different between the two cell types. There was no difference in multi-differentiational potential between 3 E-ASCs lines, however, E-ASCs had higher expression levels of chondrocyte-related genes compared to A-ASCs. These cells underwent senescence and maintained normal karyotypes.

CONCLUSIONS

Although isolated from similar adipose tissues, both types of cells displayed many contrasting characteristics. Understanding defining phenotypes of such cells is useful for making suitable choices in differing clinical indications.

Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries.

OBJECTIVE

Dental pulp stem cells (DPSCs) in permanent teeth and stem cells from human exfoliated deciduous (SHED) teeth are unique sources of mesenchymal stem cells. The aim of this study was to compare the growth characteristics and morphology of DPSCs and SHED and their immuno-phenotype using CD73 and CD146 in the 1st, 3rd, and 5th passage of cell culture.

METHODS

Growth characteristics, morphology, and colony forming efficiency were assessed for SHED and DPSCs. Immunocytochemistry and flow cytometry using CD146 and CD73 was performed for SHED and DPSCs in the 1st, 3rd and 5th passage of culture. Data was analyzed using SPSS™ software (version 17.0.0).

RESULTS

The seeding efficiency and colony forming unit efficiency was higher in SHED than in DPSCs. Flow cytometry analysis using CD73 and CD146 showed an increase in CD73 expression with increase in passage number in SHED and a decrease in CD73 expression with increase in passage number in DPSCs. There was a decrease in CD146 expression from passage one through five in SHED and DPSCs.

CONCLUSIONS

Cells isolated from the pulp of deciduous teeth and permanent teeth show difference in their growth characteristics and phenotype and are a viable source of stem cells.