Preeclampsia is a placental disease characterized by hypertension and proteinuria in pregnant women, and it is associated with a high maternal and neonatal morbidity. However, circulating biomarkers that are able to predict the prognosis of preeclampsia are lacking. Thirty-eight women were included in the current study. They consisted of 19 patients with preeclampsia (13 with severe preeclampsia and 6 with non-severe preeclampsia) and 19 gestational age-matched women with normal pregnancies as controls. We measured circulating factors that are associated with the coagulation pathway (including fibrinogen, fibronectin, factor VIII, antithrombin, protein S and protein C), endothelial activation (such as soluble endoglin and CD146), and the release of total and platelet-derived microparticles. These markers enabled us to discriminate the preeclampsia condition from a normal pregnancy but were not sufficient to distinguish severe from non-severe preeclampsia. We then used a microarray to study the transcriptional signature of blood samples. Preeclampsia patients exhibited a specific transcriptional program distinct from that of the control group of women. Interestingly, we also identified a severity-related transcriptional signature. Functional annotation of the upmodulated signature in severe preeclampsia highlighted two main functions related to "ribosome" and "complement". Finally, we identified 8 genes that were specifically upmodulated in severe preeclampsia compared with non-severe preeclampsia and the normotensive controls. Among these genes, we identified VSIG4 as a potential diagnostic marker of severe preeclampsia. The determination of this gene may improve the prognostic assessment of severe preeclampsia.

Recent studies have shown that in response to vascular damage or ischemia, bone marrow-derived endothelial progenitor cells (EPCs) are recruited into the circulation. To investigate whether antihypertensive treatment has an influence on the number of circulating EPCs, patients with essential hypertension were treated either with the angiotensin receptor antagonist telmisartan, the calcium channel blocker nisoldipine, or their combination for 6 weeks. At baseline and after 3 and 6 weeks of treatment, EPCs were identified and quantified by fluorescence-activated cell sorting (FACS) analysis and by their capacity to generate colony-forming units of the endothelial lineage (CFU-EC) in a methylcellulose-based assay. During treatment, patients in the nisoldipine groups, but not in the telmisartan group, showed a significant mobilization of EPCs, which in part had the capacity to generate large-sized colonies comprising more than 1,000 cells. Moreover, a remarkable correlation between the number of CFU-EC and the number of circulating CD133(+)/CD34(+)/CD146(+) cells was observed, thereby providing strong evidence that cells with this phenotype represent functional EPCs. No correlation was found between the numbers of CFU-EC and the blood pressure levels at any time point during the treatment. Hence, nisoldipine-induced mobilization of EPCs might represent a novel mechanism by which this antihypertensive compound independently of its blood pressure-lowering effect contributes to vasoprotection in patients with essential hypertension.

Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF-positive cells were detected within the intima and adventitia in areas where sporadic 3G5-positive pericytes were localized. Furthermore, VCAF-positive cells were identified in newly formed microvessels in association with CD34-positive/CD146-positive/c-kit-positive cells as well as in intact CD31-positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease.

The female reproductive system (FRS) has a great capacity for regeneration. The existence of somatic stem cells (SSC) that are likely to reside in distinct tissue compartments of the FRS is anticipated. Normal SSC are capable of regenerating themselves, produce a progeny of cells that differentiate and maintain tissue architecture and functional characteristics, and respond to homeostatic controls. Among those SSC of the FRS that have been identified are: a) undifferentiated cells capable of differentiating into thecal cells and synthesizing hormones upon transplantation, b) ovarian surface epithelium stem cells, mitotically responsive to ovulation, c) uterine endometrial and myometrial cells, as clonogenic epithelial and stromal cells, and d) epithelial and mesenchymal cells with self-renewal capacity and multipotential from cervical tissues. Importantly, these cells are believed to significantly contribute to the development of different pathologies and tumors of the FRS.It is now widely accepted that cancer stem cells (CSC) are at the origin of many tumors. They are capable of regenerating themselves, produce a progeny that will differentiate aberrantly and do not respond adequately to homeostatic controls. Several cell surface antigens such as CD44, CD117, CD133 and MYD88 have been used to isolate ovarian cancer stem cells. Clonogenic epithelial and stromal endometrial and myometrial cells have been found in normal and cancer tissues, as side population, label-retaining cells, and CD146/PDGF-R beta-positive cells with stem-like features. In summary, here we describe a number of studies supporting the existence of somatic stem cells in the normal tissues and cancer stem cells in tumors of the human female reproductive system.

An immortalized implantation site intermediate trophoblastic cell line, IST-1, was established from a human placenta of 7 weeks gestation. IST-1 cells phenotypically resembled the implantation site intermediate trophoblastic cells in situ and expressed Mel-CAM (MUC 18 or CD146). Mel-CAM is a cell adhesion molecule belonging to the immunoglobulin gene superfamily. It is involved in heterophilic cell-cell adhesion and plays a role in several biological processes including tumor progression. We have previously shown that Mel-CAM was highly expressed in the intermediate (extravillous) trophoblast in the human implantation site. In this study we determined the function of Mel-CAM in the interaction of trophoblast and uterine smooth muscle in the implantation site. IST-1 cells failed to adhere to immobilized recombinant Mel-CAM in solid phase whereas the uterine smooth muscle cells did. The presence of the putative Mel-CAM ligand in smooth muscle cells was further supported by the finding that Mel-CAM-transfected but not the mock-transfected U937 leukemia cells bind to the confluent monolayer of uterine smooth muscle cells. IST-1 cells attached efficiently to the monolayer of the uterine smooth muscle cells and acquired a spindle-shaped morphology simulating smooth muscle cells. The cell binding was only marginally affected by Mel-CAM blocking antibodies. However, Mel-CAM blocking antibodies and recombinant Mel-CAM promoted cell migration from IST-1 cell spheroids on the smooth muscle monolayer. Taken together, our results suggest that IST-1 cells express Mel-CAM but not the putative Mel-CAM ligand. In contrast, the uterine smooth muscle cells express the putative Mel-CAM ligand which binds to Mel-CAM on the surface of the IST-1 cells. The interaction between Mel-CAM and its putative ligand confers a stationary phenotype for trophoblastic cells. These observations are consistent with an important role for Mel-CAM in limiting trophoblastic migration within the myometrium in the implantation site.

BACKGROUND

Perivascular soft tissue tumors are relatively uncommon neoplasms of unclear line of differentiation, although most are presumed to originate from pericytes or modified perivascular cells. Among these, glomus tumor, myopericytoma, and angioleiomyoma share a spectrum of histologic findings and a perivascular growth pattern. In contrast, solitary fibrous tumor (previously termed hemangiopericytoma) was once hypothesized to have pericytic differentiation.

METHODS

Here, we systematically examine pericyte immunohistochemical markers among glomus tumor (including malignant glomus tumor), myopericytoma, angioleiomyoma, and solitary fibrous tumor. Immunohistochemical staining and semiquantification was performed using well-defined pericyte antigens, including αSMA, CD146, and PDGFRβ.

RESULTS

Glomus tumor and myopericytoma demonstrate diffuse staining for all pericyte markers, including immunohistochemical reactivity for αSMA, CD146, and PDGFRβ. Malignant glomus tumors all showed some degree of pericyte marker immunoreactivity, although it was significantly reduced. Angioleiomyoma shared a similar αSMA + CD146 + PDGFRβ+ immunophenotype; however, this was predominantly seen in the areas of perivascular tumor growth. Solitary fibrous tumors showed patchy PDGFRβ immunoreactivity only.

CONCLUSIONS

In summary, pericyte marker expression is a ubiquitous finding in glomus tumor, myopericytoma, and angioleiomyoma. Malignant glomus tumor shows a comparative reduction in pericyte marker expression, which may represent partial loss of pericytic differentiation. Pericyte markers are essentially not seen in solitary fibrous tumor. The combination of αSMA, CD146, and PDGFRβ immunohistochemical stainings may be of utility for the evaluation of pericytic differentiation in soft tissue tumors.

OBJECTIVE

Measurement of circulating endothelial cells (CECs) and progenitor cells (EPCs) has potential as a surrogate marker for monitoring anticancer treatment. This study evaluated the significance of CECs and EPCs in the blood of patients with head and neck squamous cell carcinoma.

METHODS

In a prospective trial fresh blood samples from 22 tumor patients and 18 controls were tested using multiparametric flow-cytometry. CECs were defined as CD31(+)/CD146(+) and CD45(-)/7AAD(-). EPCs were defined as CD133(+)/KDR(+) and CD3(-)/CD19(-)/CD33(-)/7AAD(-).

RESULTS

Median levels (min/max) of CECs in the tumor group were 2 (0/5) at the time of diagnosis, 1 (0/5) 1 year after therapy and 2 (0/6) in the control cohort. Median levels of EPCs were 5 (1/41) before and 10 (0/21) after treatment in the tumor group compared to 2 (0/7) in the control cohort (P < 0.001 and P = 0.03). CEC and EPC levels showed no apparent correlation with tumor size and response to radiotherapy after 18 months of observation.

CONCLUSIONS

In this pilot study CD133(+)/KDR(+) EPCs were significantly elevated in head and neck tumor patients before and after therapy. Our results warrant further studies on the use of EPCs as a surrogate marker for anticancer therapies in these patients.

In myeloid malignancies, the neoplastic clone outgrows normal hematopoietic cells toward BM failure. This event is also sustained by detrimental stromal changes, such as BM fibrosis and osteosclerosis, whose occurrence is harbinger of a dismal prognosis. We show that the matricellular protein SPARC contributes to the BM stromal response to myeloproliferation. The degree of SPARC expression in BM stromal elements, including CD146(+) mesenchymal stromal cells, correlates with the degree of stromal changes, and the severity of BM failure characterizing the prototypical myeloproliferative neoplasm primary myelofibrosis. Using Sparc(-/-) mice and BM chimeras, we demonstrate that SPARC contributes to the development of significant stromal fibrosis in a model of thrombopoietin-induced myelofibrosis. We found that SPARC deficiency in the radioresistant BM stroma compartment impairs myelofibrosis but, at the same time, associates with an enhanced reactive myeloproliferative response to thrombopoietin. The link betwen SPARC stromal deficiency and enhanced myeloid cell expansion under a myeloproliferative spur is also supported by the myeloproliferative phenotype resulting from the transplantation of defective Apc(min) mutant hematopoietic cells into Sparc(-/-) but not WT recipient BM stroma. Our results highlight a complex influence of SPARC over the stromal and hematopoietic BM response in myeloproliferative conditions.

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC's were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.

The presence of human STRO-1(+)/CD146(+) periodontal ligament (PDL) cells has been reported, but obtaining a large amount of these cells is difficult. The purpose of this study was to evaluate the percentages of STRO-1(+)/CD146(+) cells in PDL cells and determine the effects of FGF-2 on the proliferation and multilineage differentiation potency of these cells. Human PDL (HPDL) cells were individually prepared from 15 extracted teeth. HPDL cells were cultured with or without FGF-2, and the percentages of STRO-1(+)/CD146(+) cells in each HPDL cell culture was examined using FACSAria™. The STRO-1(+)/CD146(+) cells were sorted with FACSAria™, and the mRNA expression and differentiation potency of the sorted cells were subsequently examined. The numbers of the STRO-1(+)/CD146(+) cells in the FGF-2 cultures were significantly higher than those cultured in the absence of FGF-2. The sorted STRO-1(+)/CD146(+) cells expressed mRNA of PDL markers and differentiated into adipocytes and osteoblast-like cells. The present study shows that FGF-2 augmented the proliferation of the STRO-1(+)/CD146(+) cells in the HPDL cultures whilst retaining adipogenic and osteogenic differentiation potentials. Thus, it may be useful to culture HPDL cells with FGF-2 for the application of the human STRO-1(+)/CD146(+) PDL cells in periodontal tissue regeneration.

Adiponectin has antiatherogenic properties and attenuates endothelial inflammatory responses. CD146 is a novel cell adhesion molecule localized at the endothelial junction. In renal failure, endothelial dysfunction and atherosclerosis are almost universal. We studied possible correlations between adiponectin, CD146, and other markers of endothelial cell injury in patients with chronic renal failure (CRF) on conservative treatment and patients with and without diabetic nephropathy maintained on chronic ambulatory peritoneal dialysis (CAPD). We assessed adiponectin, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor (PAI-1), thrombin-activatable fibrinolysis inhibitor, and endothelial function/injury markers: von Willebrand factor, thrombomodulin, vascular cell adhesion molecule (VCAM), intercellular adhesion molecule, and CD146. Adiponectin was elevated in patients with CRF and on CAPD. It correlated significantly, with PAI-1, thrombin-activatable fibrinolysis inhibitor, intercellular adhesion molecule, VCAM, and CD146 in nondiabetics on CAPD. In diabetics, CAPD adiponectin correlated positively with C146 and VCAM and negatively with PAI and TFPI. In multivariate regression analysis, only CD146 remained a positive predictor of adiponectin in all CAPD patients. In CRF, adiponectin correlated with CD146. In healthy volunteers, adiponectin correlated with TFPI and CD146. Elevated adiponectin related to CD146 may be the expression of a counterregulatory response aimed at mitigating the consequences in endothelial damage and increased cardiovascular risk in renal failure.

BACKGROUND

Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-like-receptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile.

METHODS

Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs' incubation in basic or inflammatory medium (IL-1β, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated.

RESULTS

G-MSCs showed all stem/progenitor cells' characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test).

CONCLUSIONS

The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions.

Despite the frequent use of primary dental pulp cells in dental regenerative research, few systematic studies of stemness for osteogenic and dentinogenic differentiation of human adult pulp cells have been reported. To investigate the stemness of human adult dental pulp cells, pulp tissues were obtained from extracted third molars and used as a source of pulp cells. In FACS analysis and immunophenotyping, the general mesenchymal stem cell markers CD44, CD90, and CD146 were highly expressed in early passages of the pulp cell culture. The stem cell population was dramatically decreased in an expansion culture of human dental pulp cells. When pulp cells were treated with additives such as β-glycerophosphate, ascorbic acid, and dexamethasone, nodule formation was facilitated and mineralization occurred within 2 weeks. Expression of osteogenic markers such as alkaline phosphatase, osteocalcin, and osteonectin was relatively low in undifferentiated cells, but increased significantly under differentiation conditions in whole passages. Dentinogenic markers such as dentin sialophosphoprotein and dentin matrix protein-1 appeared to decrease in their expression with increasing passage number; however, peak levels of expression occurred at around passage 5. These data suggested that stem cells with differentiation potential might exist in the dental pulp primary culture, and that their phenotypes were changed during expansion culture over 8-9 passages. Under these conditions, a dentinogenic population of pulp cells occurred in limited early passages, whereas osteogenic cells occurred throughout the whole passage range.

BACKGROUND

Human dental pulp stem/progenitor cells (DPSCs) show remarkable regenerative potential in vivo. During regeneration, DPSCs may interact with their inflammatory environment via toll-like receptors (TLRs). The present study aimed to depict for the first time the TLR expression profile of DPSCs.

METHODS

Cells were isolated from human dental pulp, STRO-1-immunomagnetically sorted, and seeded out to obtain single colony-forming units. DPSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, and CD146 expression and for their multilineage differentiation potential. After incubation of DPSCs in basic or inflammatory medium (interleukin-1β, interferon-γ, interferon-α, tumor necrosis factor-α), TLR expression profiles were generated (DPSCs and DPSCs-i).

RESULTS

DPSCs showed all characteristics of stem/progenitor cells. In basic medium DPSCs expressed TLRs 1-10 in different quantities. The inflammatory medium upregulated the expression of TLRs 2, 3, 4, 5, and 8, downregulated TLRs 1, 7, 9, and 10, and abolished TLR6.

CONCLUSIONS

The current study describes for the first time the distinctive TLR expression profile of DPSCs in uninflamed and inflamed conditions.

The soluble form of CD146 has been reported to be present in various inflammatory diseases and displays pro-inflammatory properties. However, little is known about sCD146 in multiple sclerosis (MS). Here we show that sCD146 is significantly elevated in the cerebrospinal fluid of patients with active MS compared with that of inactive MS or patients with non-demyelinating diseases. Moreover, abnormally increased sCD146 in the CSF of active MS patients correlated with albumin quotient, MBP antibody and MOG antibody from both CSF and sera. Importantly, the level of CSF sCD146 is correlated with levels of inflammatory factors, such as TNFα, IFNγ, IL-2, and IL-17A in the CSF. We also found that CSF sCD146 might originate from membrane-bound CD146 on inflamed blood-brain barrier (BBB) endothelial cells. In addition, sCD146 promotes leukocyte transmigration in vitro, at least in part by stimulating the expression of ICAM-1 and VCAM-1 on endothelial cells. Our findings suggest that CSF levels of sCD146 may provide a potential marker for monitoring disease activity in MS patients.

Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures.

OBJECTIVE

Periodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146-PDLCs).

METHODS

CD146±PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146±PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration).

RESULTS

CD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146-PDLCs. TNF-α at a dose of 2.5ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146-PDLCs. At 10ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146-PDLCs was not altered by TNF-α.

CONCLUSIONS

CD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146-PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146-PDLCs were found to be less sensitive to TNF-α.

Characterization of cells is important for facilitating cell-based therapies for degenerative diseases of intervertebral discs. For this purpose, we analyzed mouse annulus fibrosus cells by flowcytometory to detect phenotypic change in their primary cultures. After examination of sixteen cell surface proteins, we focused on CD146 that solely increased during culture expansion. CD146 is known to be a marker for mesenchymal stem cells and for their vascular smooth muscle commitment with expression of contractile phenotype enhanced by SM22α. We sorted CD146+ cells to elucidate their characteristics and the key factors that play a role in this change. Whole cell cultures showed the ability for tripotent differentiation toward mesenchymal lineages, whereas sorted CD146+ cells did not. Expression of CD146 was elevated by addition of transforming growth factor β1, and sorted CD146+ cells expressed higher levels of mRNA for SM22α and Elastin than did CD146- cells. Morphologically, CD146+ cells more broadly deposited extracellular type I collagen than CD146- cells and showed filamentous actin bundles traversing their cytoplasm and cell-cell junctions. Moreover, CD146+ cells demonstrated significantly higher gel contraction properties than CD146- cells when they were embedded in collagen gels. Human annulus fibrosus CD146+ cells also showed higher contractility. Immunohistochemistry determined CD146+ cells localized to the outermost annulus layers of mouse intervertebral disc tissue with co-expression of SM22α. These results suggest that increment of CD146 expression indicates gradual change of cultured annulus fibrosus cells to express a contractile phenotype and that transforming growth factor β1 enhances this cellular commitment. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1361-1372, 2016.

BACKGROUND

We investigated the extent, modalities and reversibility of changes at cellular level in the expression of genes and proteins occurring upon Hind limb unloading (HU) in the tibiae of young C57BL/6J male mice. We focused on the effects of HU in chondrogenic, osteogenic, and marrow mesenchymal cells.

METHODS

We analyzed for expression of genes and proteins at two time points after HU (7 and 14 days), and at 14 days after recovery from HU. Levels of mRNAs were tested by in situ hybridization. Protein levels were tested by immunohistochemistry. We studied genes involved in osteogenesis (alkaline phosphatase (AP), osteocalcin (OC), bonesialoprotein (BSP), membrane type1 matrix metalloproteinase (MT1-MMP)), in extracellular matrix (ECM) formation (procollagenases (BMP1), procollagenase enhancer proteins (PCOLCE)) and remodeling (metalloproteinase-9 (MMP9), RECK), and in bone homeostasis (Stro-1, CXCL12, CXCR4, CD146).

RESULTS

We report the following patterns and timing of changes in gene expression induced by HU: 1) transient or stable down modulations of differentiation-associated genes (AP, OC), genes of matrix formation, maturation and remodelling, (BMP1, PCOLCEs MMP9) in osteogenic, chondrogenic and bone marrow cells; 2) up modulation of MT1-MMP in these same cells, and uncoupling of its expression from that of AP; 3) transient down modulation of the osteoblast specific expression of BSP; 4) for genes involved in bone homeostasis, up modulation in bone marrow cells at distal epiphysis for CXCR4, down modulation of CXCL12, and transient increases in osteoblasts and marrow cells for Stro1. 14 days after limb reloading expression returned to control levels for most genes and proteins in most cell types, except AP in all cells, and CXCL12, only in bone marrow.

CONCLUSIONS

HU induces the coordinated modulation of gene expression in different mesenchymal cell types and microenvironments of tibia. HU also induces specific patterns of expression for homeostasis related genes and modulation of mRNAs and proteins for ECM deposition, maturation and remodeling which may be key factors for bone maintenance.

BACKGROUND

Optimization of fat grafting continues to gain increasing attention in the field of regenerative medicine. "Nanofat grafting" implements mechanical emulsification and injection of standard lipoaspirate for the correction of superficial rhytides and skin discoloration; however, little is known about the cellular constituents of the graft. Based on recent evidence that various stressors can induce progenitor activity, the authors hypothesized that the shear forces used in common fat grafting techniques may impact their regenerative capacities.

METHODS

Lipoaspirates were obtained from 10 patients undergoing elective procedures. Half of each sample was subjected to nanofat processing; the other half was left unchallenged. The viscosity of each sample was measured for computational analysis. The stromal vascular fraction of each sample was isolated, quantified, and analyzed by means of flow cytometry with two multicolor fluorescence antibody panels.

RESULTS

Standard lipoaspirate is ideally suited for mechanical stress induction. The mechanical emulsification involved in nanofat processing did not affect cell number; however, viability was greatly reduced when compared with the stromal vascular fraction of standard lipoaspirate. Interestingly, nanofat processing resulted in stress-induced stromal vascular fraction with a higher proportion of endothelial progenitor cells, mesenchymal stem cells, and multilineage differentiating stress-enduring cells. Single-parameter analysis also revealed significant increases in CD34, CD13, CD73, and CD146 of the stress-induced stromal vascular fraction, markers associated with mesenchymal stem cell activity.

CONCLUSIONS

Mechanical processing used in techniques such as nanofat grafting induces the up-regulation of progenitor phenotypes consistent with multipotency and pluripotency. These data provide a first step in characterizing the potential regenerative benefits realized through stress induction in fat grafting.

UNASSIGNED

Therapeutic, V.

BACKGROUND

Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in recent times as involved in cell-to-cell communication between distant cells. Nevertheless, TNT-like, long filopodial processes had already been described in the last century as connecting facing, growing microvessels during the process of cerebral cortex vascularization and collateralization. Here we have investigated the possible presence and the cellular origin of TNTs during normal brain vascularization and also in highly vascularized brain tumors.

METHODS

We searched for TNTs by high-resolution immunofluorescence confocal microscopy, applied to the analysis of 20-µm, thick sections from lightly fixed, unembedded samples of both developing cerebral cortex and human glioblastoma (GB), immunolabeled for endothelial, pericyte, and astrocyte markers, and vessel basal lamina molecules.

RESULTS

The results revealed the existence of pericyte-derived TNTs, labeled by proteoglycan NG2/CSPG4 and CD146. In agreement with the described heterogeneity of these nanostructures, ultra-long >> 300 µm) and very thin (< 0.8 µm) TNTs were observed to bridge the gap between the wall of distant vessels, or were detected as short (< 300 µm) bridging cables connecting a vessel sprout with its facing vessel or two apposed vessel sprouts. The pericyte origin of TNTs ex vivo in fetal cortex and GB was confirmed by in vitro analysis of brain pericytes, which were able to form and remained connected by typical TNT structures.

CONCLUSIONS

None of the multiple roles described for TNTs can be excluded from a possible involvement during the processes of both normal and pathological vessel growth. A possible function, suggested by the pioneering studies made during cerebral cortex vascularization, is in cell searching and cell-to-cell recognition during the processes of vessel collateralization and vascular network formation. According to our results, it is definitely the pericyte-derived TNTs that seem to actively explore the surrounding microenvironment, searching for (site-to-site recognition), and connecting with (pericyte-to-pericyte and/or pericyte-to-endothelial cell communication), the targeted vessels. This idea implies that TNTs may have a primary role in the very early phases of both physiological and tumor angiogenesis in the brain.

OBJECTIVE

The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells.

METHODS

Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4),β-glycerophosphate and L-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5mM) and TEGDMA (0.05-0.25mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2mM) and TEGDMA (1mM) were also evaluated.

RESULTS

DTSCs cultures were positive for STRO-1 (7.53±2.5%), CD146 (91.79±5.41%), CD34 (11.87±3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated.

CONCLUSIONS

These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.

Human adipose-derived stem cell populations express cell surface markers such as CD105, CD73, CD146 and CD140a/PDFGRα. However, it was unclear whether these markers could discriminate subpopulations of undifferentiated cells and whether the expression of these markers is modulated during differentiation. To address this issue, we analysed the immunophenotype of cultured human multipotent adipose derived stem (hMADS) cell populations at different adipocyte differentiation steps. We found that 100% of undifferentiated cells expressed CD73 and CD105. In contrast, CD146 and CD140a/PDFGRα marked two different subpopulations of cells. CD140a/PDGFRα subpopulation was regulated by FGF2, a critical factor of human adipose-derived stem cell self-renewal. During differentiation, CD73 was maintained and marked lipid-laden cells, whereas CD105 expression was inhibited in fully differentiated cells. The percentage of CD146 and CD140a/PDFGRα-positive cells declined as soon as cells had undergone differentiation. Altogether, these data support the notion that expanded adipose-derived stem cells are heterogeneous mixtures of cells and cell surface markers studied can discriminate subpopulations.

CD146 (MUC-18, MCAM) expression on cancer cells correlates with cancer progression and a bad prognosis in several tumors, including melanoma and pancreatic tumors. Deciphering the mechanism mediating the CD146 role in cancer is essential for generating new therapeutic strategies. We found that CD146 expression in cancer cells is associated with a secretion of soluble CD146 (sCD146) that constitutes an active player in tumor development. Indeed, sCD146 induces the overexpression of its binding protein, angiomotin, on both endothelial and cancer cells and promotes both paracrine effects on angiogenesis and autocrine effects on cancer cells proliferation and survival. These last effects are mediated in part through the induction and phosphorylation of c-myc in cancer cells. In mice models xenografted with human CD146-positive melanoma or pancreatic cancer cells, administration of a novel monoclonal antibody specifically targeting sCD146, but not its membrane form, successfully suppresses tumor vascularization and growth. Our findings demonstrate that sCD146 secreted by CD146-positive tumors mediates important pro-angiogenic and pro-tumoral effects. Targeting sCD146 with a novel neutralizing antibody could thus constitute an innovative therapeutic strategy for the treatment of CD146-positive tumors.