Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Mesenchymal stromal cells (MSCs) are multipotent cells that can be differentiated in vitro into a variety of cell types, including adipocytes or osteoblasts. Our recent studies indicated that a high expression of CD146 on MSCs from bone marrow correlates with their robust osteogenic differentiation potential. We therefore investigated if expression of CD146 on MSCs from the placenta correlates with a similar osteogenic differentiation potential. The MSCs were isolated specifically from the endometrial and fetal parts of human term placenta and expanded in separate cultures and compared with MSCs from bone marrow as controls. The expression of cell surface antigens was investigated by flow cytometry. Differentiation of MSCs was documented by cytochemistry and analysis of typical lineage marker genes. CD146-positive MSCs were separated from CD146-negative cells by magnet-assisted cell sorts (MACS). We report that the expression of CD146 is associated with a higher osteogenic differentiation potential in human placenta-derived MSCs (pMSCs) and the CD146(pos) pMSCs generated a mineralized extracellular matrix, whereas the CD146(neg) pMSCs failed to do so. In contrast, adipogenic and chondrogenic differentiation of pMSCs was not different in CD146(pos) compared with CD146(neg) pMSCs. Upon enrichment of pMSCs by MACS, the CD146(neg) and CD146(pos) populations maintained their expression levels for this antigen for several passages in vitro. We conclude that CD146(pos) pMSCs either respond to osteogenic stimuli more vividly or, alternatively, CD146(pos) pMSCs present a pMSC subset that is predetermined to differentiate into osteoblasts.

In ischemic acute kidney injury, renal blood flow is decreased. We have previously shown that reperfused, transplanted kidneys exhibited ischemic injury to vascular endothelium and that preservation of peritubular capillary endothelial integrity may be critical to recovery from ischemic injury. We hypothesized that bone marrow-derived (BMD) endothelial progenitor cells (EPCs) might play an important role in renal functional recovery after ischemia. We tested this hypothesis in recipients of cadaveric renal allografts before and for 2 weeks after transplantation. We found that the numbers of circulating CD34-positive EPCs and CD146-positive endothelial cells (ECs) decreased immediately after ischemia-reperfusion. In renal allograft tissues obtained 1 hr after reperfusion, CD34-positive cells were more frequently observed along the endothelial lining of peritubular capillaries compared with non-ischemic controls. Moreover, 0-17.5% of peritubular capillary ECs were of recipient origin. In contrast, only 0.1-0.7% of tubule cells were of recipient origin. Repeat graft biopsy samples obtained 35 and 73 days after transplant did not contain capillary ECs of recipient origin, whereas 1.4% and 12.1% of tubule cells, respectively, were of recipient origin. These findings suggest that BMD EPCs and ECs may contribute to endothelial repair immediately after ischemia-reperfusion.

BACKGROUND

We investigated whether circulating endothelial cells (CECs) predict clinical outcome of first-line chemotherapy and bevacizumab in metastatic colorectal cancer (mCRC) patients.

METHODS

In a substudy of the randomized phase II FNCLCC ACCORD 13/0503 trial, CECs (CD45- CD31+ CD146+ 7-amino-actinomycin- cells) were enumerated in 99 patients by four-color flow cytometry at baseline and after one cycle of treatment. We correlated CEC levels with objective response rate (ORR), 6-month progression-free survival (PFS) rate (primary end point of the trial), PFS, and overall survival (OS). Multivariate analyses of potential prognostic factors, including CEC counts and Köhne score, were carried out.

RESULTS

By multivariate analysis, high baseline CEC levels were the only independent prognostic factor for 6-month PFS rate (P < 0.01) and were independently associated with worse PFS (P = 0.02). High CEC levels after one cycle were the only independent prognostic factor for ORR (P = 0.03). High CEC levels at both time points independently predicted worse ORR (P = 0.025), 6-month PFS rate (P = 0.007), and PFS (P = 0.02). Köhne score was the only variable associated with OS.

CONCLUSIONS

CEC levels at baseline and after one treatment cycle may independently predict ORR and PFS in mCRC patients starting first-line bevacizumab and chemotherapy.

The persistence of cholesterol-engorged macrophages (foam cells) in the artery wall fuels the development of atherosclerosis. However, the mechanism that regulates the formation of macrophage foam cells and impedes their emigration out of inflamed plaques is still elusive. Here, we report that adhesion receptor CD146 controls the formation of macrophage foam cells and their retention within the plaque during atherosclerosis exacerbation. CD146 is expressed on the macrophages in human and mouse atheroma and can be upregulated by oxidized low-density lipoprotein (oxLDL). CD146 triggers macrophage activation by driving the internalization of scavenger receptor CD36 during lipid uptake. In response to oxLDL, macrophages show reduced migratory capacity toward chemokines CCL19 and CCL21; this capacity can be restored by blocking CD146. Genetic deletion of macrophagic CD146 or targeting of CD146 with an antibody result in much less complex plaques in high-fat diet-fed ApoE-/- mice by causing lipid-loaded macrophages to leave plaques. Collectively, our findings identify CD146 as a novel retention signal that traps macrophages within the artery wall, and a promising therapeutic target in atherosclerosis treatment.

OBJECTIVE

Angiogenesis and lymphangiogenesis are important steps in tumor growth and dissemination and are of prognostic importance in solid tumors. The determination of microvessel density (MVD) by immunohistology is subject to considerable variability between different laboratories and observers. We compared MVD determination by immunohistology and quantitative real-time PCR and correlated the results with clinical variables.

METHODS

The expression of endothelial antigens vascular endothelial cadherin (CD144), P1H12 (CD146), tie-2, and VEGFR-2, and lymphatic endothelial markers VEGFR-3, Prox, and LYVE was assessed by quantitative PCR (qPCR) in primary surgical samples. The expression of angiogenetic growth factors VEGF-A, VEGF-C, VEGF-D, angiopoietin-1, and angiopoietin-2 was quantified by PCR and correlated with MVD and clinical variables.

RESULTS

The expression of endothelial antigens vascular endothelial cadherin (CD144), P1H12 (CD146), tie-2, and VEGFR-2 correlated with each other in 54 samples of primary esophageal cancer (P < 0.0001 for all comparisons). MVD determined immunohistologically by CD31 staining in a subgroup of 35 patients correlated significantly with the qPCR method. The expression of angiogenetic growth factors VEGF-A, VEGF-C, VEGF-D, angiopoietin-1, and angiopoietin-2 was significantly associated with MVD (P < 0.0001 for all comparisons). Analysis of the expression of lymphendothelial markers VEGFR-3, Prox, and LYVE revealed concordant results, indicating that quantification of lymphendothelial cells is possible by qPCR. The presence of lymph node metastasis on surgical specimens was significantly correlated with MVD (P < 0.003), VEGFR-2 (P < 0.048), and VEGF-C (P < 0.042) expression.

CONCLUSIONS

These results indicate that quantification of MVD by qPCR in surgical samples of esophageal carcinoma yields similar results with immunohistology. Interestingly, the extent of angiogenesis and lymphangiogenesis was not related in individual tumor samples. Lymph node metastases could be predicted by MVD and VEGF-C expression.

Human dental pulp derived from exfoliated deciduous teeth has been described as a promising alternative source of multipotent stem cells. While these cells share certain similarities with mesenchymal stem-like cells (MSC) isolated from other tissues, basically they are still poorly characterized. In this study, for the first time, a proteomic map of abundantly expressed proteins in stromal cells derived from the dental pulp of human exfoliated deciduous teeth (SHED) was established. We also analyzed proteomic signatures of 2 clonal strains derived from SHEDs by single-cell cloning. The SHEDs were established from enzyme-disaggregated deciduous dental pulp from 6-year-old children. They had typical fibroblastoid morphology and high colony-forming efficiency index (16.4%). Cloning was performed at the second passage using limiting dilution in a 96-well plate (0.3 cell/well). Differentiation assessment revealed strong osteogenic but no adipogenic potential of the SHEDs in either clonal strain. The cells expressed characteristic antigens of MSC-like cells, including CD73, CD90, CD105, CD146, and did not express hematopoietic markers CD14, CD34, and CD45, as assessed with FACS analysis. For proteomic studies, cytosolic and nuclear proteins were analyzed with 2-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization (MALDI)-time of fl ight (TOF)-mass spectrometry (MS). All proteins were identified with high level of confidence (the lowest sequence coverage was 27%). Identification of highly expressed proteins in SHEDs revealed proteomic profiles very similar to that of MSC-like cells derived from other tissues. We also found a high degree of similarity between proteomic signatures of primary SHEDs and clonal cell strains. Thus, our data confirm a close resemblance between SHEDs and MSC-like cells from other tissues and may serve as starting point for creating-comprehensive proteomic maps.

Chronic intermittent hypoxia (CIH) inhibits plasma lipoprotein clearance and adipose lipoprotein lipase (LPL) activity in association with upregulation of an LPL inhibitor angiopoietin-like protein 4 (Angptl4). We hypothesize that CIH inhibits triglyceride (TG) uptake via Angptl4 and that an anti-Angptl4-neutralizing antibody would abolish the effects of CIH. Male C57BL/6J mice were exposed to four weeks of CIH or intermittent air (IA) while treated with Ab (30 mg/kg ip once a week). TG clearance was assessed by [H(3)]triolein administration retroorbitally. CIH delayed TG clearance and suppressed TG uptake and LPL activity in all white adipose tissue depots, brown adipose tissue, and lungs, whereas heart, liver, and spleen were not affected. CD146+ CD11b- pulmonary microvascular endothelial cells were responsible for TG uptake in the lungs and its inhibition by CIH. Antibody to Angptl4 decreased plasma TG levels and increased TG clearance and uptake into adipose tissue and lungs in both control and CIH mice to a similar extent, but did not reverse the effects of CIH. The antibody reversed the effects of CIH on LPL in adipose tissue and lungs. In conclusion, CIH inactivates LPL by upregulating Angptl4, but inhibition of TG uptake occurs predominantly via an Angptl4/LPL-independent mechanism.

BACKGROUND

Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization.

RESULTS

Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts.

CONCLUSIONS

Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature.

About 30 years ago circulating endothelial cells (CEC) were first observed in peripheral blood. Since then CEC have been established as a reliable indicator of vascular injury and damage and more sophisticated detection techniques, such as immunomagnetic isolation and fluorescence-activated cell sorting (FACS), have become available. However even today there remains controversy as to the best approach to isolate and enumerate these cells. Here, we review the isolation and enumeration of CEC with an emphasis on CD146-driven immunomagnetic isolation and FACS as the two competing techniques. We describe advantages and pitfalls of both approaches. Moreover, we provide a list of clinical studies in this field and describe the possible clinical utility of CEC as a surrogate marker for vascular damage and dysfunction. In addition, we review the phenotype of CEC and discuss mechanisms of detachment. Recent evidence has also revealed interesting interactions between CEC and healthy endothelium in vitro although the relevance of these findings for human vascular disease in vivo remains unclear. Finally, we highlight differences between circulating endothelial cells and endothelial progenitor cells. In summary, CEC must be regarded as a sensitive and specific marker of endothelial damage as well as a potential mediator in vascular disease.

To determine whether exercise increases endothelial progenitor cells (EPCs) in patients with peripheral vascular disease, we developed a multi-parameter flow cytometry assay to rigorously assess EPCs and mature endothelial cells (ECs) in control subjects and patients with peripheral artery disease (PAD) subjected to graded exercise. Blood was collected from young healthy subjects (n = 9, mean age 33 years), older healthy subjects (n = 13, mean age 66 years), and older subjects with PAD (n = 15, mean age 69 years) before and 10 minutes after exercise. White blood cells were isolated and stained with a five-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33, PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion. Viable, low, side scatter singlets that were CD3-, 19-, and 33-negative were counted. While baseline levels of EPCs and ECs were similar among all subjects, young healthy subjects demonstrated significantly greater (p < 0.05) levels of progenitor cells (PCs) than older healthy and PAD subjects. Levels of EPCs and ECs tended to increase in all subjects after exercise; however, increases in PCs were only observed in young healthy and PAD subjects. Further, trends in the magnitude of change of subsets with exercise were most similar between young and PAD subjects. Our findings suggest that aging may reduce baseline circulating levels of PCs, but not EPCs or ECs, and that exercise-induced mobilization of subsets may differ depending on age and presence of PAD.

OBJECTIVE

Mesenchymal stem cells are an interesting source of material for regenerative medicine. The present study aimed at characterizing the phenotype and differentiation potential of adherent synovial fluid-derived cells from temporomandibular joint (TMJ) disorder patients.

METHODS

Synovial fluid collection takes place during TMJ cavity irrigation arthrocentesis under local anesthesia. The synovial fluid-derived adherent cells were fibroblast-like and spindle-shaped. Ex vivo-expanded synovial fluid-derived cells were shown to express STRO-1 and CD146, previously found to be present in bone marrow mesenchymal stem cells. Further, they were identified as being capable of differentiating into a variety of cell types including osteoblasts, chondrocytes, adipocytes, and neurons.

RESULTS

The present study demonstrates that human pluripotent cells can be isolated from synovial fluid. These synovial fluid-derived cells cannot only be derived from a very accessible resource, but are also capable of providing sufficient cells for potential clinical applications.

CONCLUSIONS

These cells may play a role in the regenerative response during arthritic diseases and are promising candidates for developing novel cell-based therapeutic approaches for postnatal skeletal tissue repair.

Placenta has attracted increasing attention over the past decade as a stem cell source for regenerative medicine. In particular, the amniochorionic membrane has been shown to harbor populations of mesenchymal stromal cells (MSCs). In this study, we have characterized ex vivo expanded MSCs from the human amniotic (hAMSCs) and chorionic (hCMSCs) membranes of human full-term placentas and adult bone marrow (hBMSCs). Our results show that hAMSCs, hCMSCs, and hBMSCs express typical mesenchymal (CD73, CD90, CD105, CD44, CD146, CD166) and pluripotent (Oct-4, Sox2, Nanog, Lin28, and Klf4) markers but not hematopoietic markers (CD45, CD34). Ex vivo expanded hAMSCs were found to be of fetal origin, while hCMSCs cultures contained only maternal cells. Cell proliferation was significantly higher in hCMSCs, compared to hAMSCs and hBMSCs. Integrin profiling revealed marked differences in the expression of α subunits between the three cell sources. Cadherin receptors were consistently expressed on a subset of progenitors (ranging from 1% to 60%), while N-CAM (CD56) was only expressed in hAMSCs and hCMSCs but not in hBMSCs. When induced to differentiate, hAMSCs and hCMSCs displayed strong chondrogenic and osteogenic differentiation potential but very limited capacity for adipogenic conversion. In contrast, hBMSCs showed strong differentiation potential along the three lineages. These results illustrate how MSCs from different ontological sources display differential expression of cell-fate mediators and mesodermal differentiation capacity.

Increased numbers of CD146-bearing circulating endothelial cells (CECs) in the peripheral blood probably represent the most direct evidence of endothelial cell damage. As acute ischaemic strokes are associated with endothelial abnormalities, we hypothesised that these CECs are raised in acute stroke, and that they would correlate with the other indices of endothelial perturbation, i.e. plasma von Willebrand factor (vWf) and soluble E-selectin. We studied 29 hypertensive patients (19 male; mean age 63 years) who presented with an acute stroke and compared them with 30 high risk hypertensive patients (21 male; mean age 62 years) and 30 normotensive controls (16 male; mean age 58 years). CECs were estimated by CD146 immunobead capture, vWf and soluble E-selectin by ELISA. Patients with an acute ischaemic stroke had significantly higher numbers of CECs/ml of blood (p<0.001) plasma vWf (p=0.008) soluble E-selectin (p=0.002) and higher systolic blood pressure (SBP) as compared to the other groups. The number of CECs significantly correlated with soluble E-selectin (r=0.432, p<0.001) and vWf (r=0.349, p=0.001) but not with SBP (r=0.198, p=0.069). However, in multivariate analysis, only disease group (i.e. health, hypertension or stroke) was associated with increased CECs. Acute ischaemic stroke is associated with increased numbers of CECs. The latter correlate well with established plasma markers of endothelial dysfunction or damage, thus unequivocally confirming severe vasculopathy in this condition. However, the greatest influence on CECs numbers was clinical group.

BACKGROUND

Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition, numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD), we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs), EPCs, and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset.

METHODS

Blood was collected from young healthy subjects (N = 9, mean age 33 +/- 8 years), older healthy subjects (N = 13, mean age 66 +/- 8 years), and older subjects with PAD (N = 15, mean age 69 +/- 8 years). After ammonium chloride lysis, cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33 (lineage panel), PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion, and only viable, low to medium side scatter lineage-negative singlets were analyzed. In some studies, cells were sorted for morphological studies. Subsets were defined as indicated later.

RESULTS

Our results, using a comprehensive flow cytometric panel, indicate that CD133+, CD34+, and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals, both healthy and with PAD. However, the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings.

CONCLUSIONS

We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs, but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status.

White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

BACKGROUND

A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications.

RESULTS

Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion.

CONCLUSIONS

This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications.

Recent studies have identified a new human dental derived progenitor cell population with multi-lineage differentiation potential referred to as human periapical cyst mesenchymal stem cells (hPCy-MSCs). In the present study, we compared two subpopulations of hPCy-MSCs characterised by the low or high expression of CD146 to establish whether this expression can regulate their stem cell properties. Using flow cytometry, we evaluated the stem cell marker profile of hPCy-MSCs during passaging. Furthermore, CD146Low and CD146High cells were sorted by magnetic beads and subsequently both cell populations were evaluated for differences in their proliferation, self-renewal, stem cell surface markers, stemness genes expression and osteogenic differentiation potential.We found that hPCy-MSCs possessed a stable expression of several mesenchymal stem cell surface markers, whereas CD146 expression declined during passaging.In addition, sorted CD146Low cells proliferated significantly faster, displayed higher colony-forming unit-fibroblast capacity and showed higher expression of Klf4 when compared to the CD146High subset. Significantly, the osteogenic potential of hPCy-MSCs was greater in the CD146Low than in CD146High population. These results demonstrate that CD146 is spontaneously downregulated with passaging at both mRNA and protein levels and that the high expression of CD146 reduces the proliferative, self-renewal and osteogenic differentiation potential of hPCy-MSCs. In conclusion, our study demonstrates that changes in the expression of CD146 can influence the stem cell properties of hPCy-MSCs.

As well as systemic vascular endothelial cells, the liver has specialised sinusoidal endothelial cells (LSEC). LSEC dysfunction has been documented in many diseased states yet their phenotype in normal human liver has not been comprehensively assessed. Our aim was to improve characterisation of subsets of endothelial cells and associated pericytes in the human liver. Immunofluorescence microscopy was performed on normal human liver tissue samples to assess endothelial and structural proteins in a minimum of three donors. LSEC are distributed in an acinar pattern and universally express CD36, but two distinctive subsets of LSEC can be identified in different acinar zones. Type 1 LSEC are CD36hiCD32-CD14-LYVE-1- and are located in acinar zone 1 of the lobule, while Type 2 LSEC are LYVE-1+CD32hiCD14+CD54+CD36mid-lo and are located in acinar zones 2 and 3 of the lobule. Portal tracts and central veins can be identified using markers for systemic vascular endothelia and pericytes, none of which are expressed by LSEC. In areas of low hydrostatic pressure LSEC are lined by stellate cells that express the pericyte marker CD146. Our findings identify distinctive populations of LSEC and distinguish these cells from adjacent stellate cells, systemic vasculature and pericytes in different zones of the liver acinus.

Abstract CD146 (MUC18, Mel-CAM/MCAM) is a transmembrane protein, originally identified as a biomarker of melanoma, and plays an important role in cancer invasion and metastasis. Further studies revealed that CD146 as a novel endothelial marker was also involved in angiogenesis. Previous studies reported several anti-CD146 antibodies, such as MUC18, A32, S-endo1, and P1H12, showing different binding patterns to the endothelium of various types of blood vessels. To examine the possibility that antibodies targeting different epitopes on CD146 could have different behaviors, we generated a panel of anti-human CD146 monoclonal antibodies, named AA1-5 and AA7, by immunizing mice with human CD146 protein purified from HUVEC. Their specificity and binding affinity were intensively characterized using Western blotting, flow cytometry, and immunohistochemical assay. On the basis of epitope mapping, we divided the six monoclonal antibodies (MAb) into two groups, groups V1 and C2-2, corresponding to the different extracellular domains harboring these epitopes, the first IgV and the second IgC2 domains, respectively. Furthermore, owing to different epitopes, the two groups of antibodies behaved differentially in cellular and histological levels. Therefore, these anti-CD146 MAbs targeting different domains should be useful tools in studying the expression and function of human CD146.

BACKGROUND

The osteoanabolic properties of PTH may be due to increases in the number and maturity of circulating osteogenic cells. Hypoparathyroidism is a useful clinical model because this hypothesis can be tested by administering PTH.

OBJECTIVE

The objective of the study was to characterize circulating osteogenic cells in hypoparathyroid subjects during 12 months of PTH (1-84) administration.

METHODS

Osteogenic cells were characterized using flow cytometry and antibodies against osteocalcin, an osteoblast-specific protein product, and stem cell markers CD34 and CD146. Changes in bone formation from biochemical markers and quadruple-labeled transiliac crest bone biopsies (0 and 3 month time points) were correlated with measurements of circulating osteogenic cells.

METHODS

The study was conducted at a clinical research center.

METHODS

Nineteen control and 19 hypoparathyroid patients were included in the study.

METHODS

Intervention included the administration of PTH (1-84).

RESULTS

Osteocalcin-positive cells were lower in hypoparathyroid subjects than controls (0.7 ± 0.1 vs. 2.0 ± 0.1%; P < 0.0001), with greater coexpression of the early cell markers CD34 and CD146 among the osteocalcin-positive cells in the hypoparathyroid subjects (11.0 ± 1.0 vs. 5.6 ± 0.7%; P < 0.001). With PTH (1-84) administration, the number of osteogenic cells increased 3-fold (P < 0.0001), whereas the coexpression of the early cell markers CD34 and CD146 decreased. Increases in osteogenic cells correlated with circulating and histomorphometric indices of osteoblast function: N-terminal propeptide of type I procollagen (R(2) = 0.4, P ≤ 0.001), bone-specific alkaline phosphatase (R(2) = 0.3, P < 0.001), osteocalcin (R(2) = 0.4, P < 0.001), mineralized perimeter (R(2) = 0.5, P < 0.001), mineral apposition rate (R(2) = 0.4, P = 0.003), and bone formation rate (R(2) = 0.5, P < 0.001).

CONCLUSIONS

It is likely that PTH stimulates bone formation by stimulating osteoblast development and maturation. Correlations between circulating osteogenic cells and histomorphometric indices of bone formation establish that osteoblast activity is being identified by this methodology.

The lifespan of the tooth is influenced by the periodontal ligament (PDL), a specialized connective tissue that connects the cementum with the tooth socket bone. Generation of a cell line from PDL progenitor/stem cells would allow development of tissue engineering-based regenerative PDL therapy. However, little is known about the characteristics of PDL progenitor/stem cells because PDL tissue consists of a heterogeneous cell population and there are no pure PDL cell lines. Recently, we succeeded in immortalizing primary human PDL fibroblasts (HPLFs) by transfecting them with SV40 T-antigen and hTERT (Cell Tissue Res 2006; 324: 117-125). In this study, we isolated three clonal cell lines from these immortalized cells (lines 1-4, 1-11, and 1-24) that express RUNX-2, Col I, ALP, OPN, OCN, RANKL, OPG, scleraxis, periostin, Col XII, and alpha-SMA mRNA. Immunocytochemical analysis demonstrated that CD146 was expressed in cell lines 1-4 and 1-11 and that STRO-1 was expressed in lines 1-11 and 1-24. Lines 1-4 and 1-11 differentiated into osteoblastic cells and adipocytes when cultured in lineage-specific differentiation media. Four weeks after transplanting cell line 1-11 into immunodeficient mice with beta-tricalcium phosphate (beta-TCP), the transplant produced cementum/bone-like tissues around the beta-TCP. Eight weeks after transplantation, the 1-11 cell transplant formed PDL-like structures on the surface of the beta-TCP. These data suggest that cell line 1-11 was derived from a progenitor/stem cell present in the PDL and should be very useful for studying the biology and regeneration of human periodontium.

Dental follicle cells (DFCs) are a heterogeneous population that exhibit a variety of phenotypes. However, it remains unclear whether DFCs can maintain stem cell characteristics, or mediate tissue-regeneration to form single or complex tissues in the periodontium, after long-term culturing. Therefore, DFCs were isolated from human impacted molars (HIM-DFCs), passaged >30 times, and then evaluated for their heterogeneity and multipotential differentiation. Morphology, proliferation, epitope profile, and mineralization characteristics of clones derived from single HIM-DFCs in vitro were also assayed. HIM-DFCs (passage #30) were found to be positive for the heterogeneous markers, Notch-1, stro-1, alkaline phosphomonoesterase (ALP), type I collagen (COL-I), type III collagen (COL-III), and osteocalcine. Moreover, passage #30 of the HDF1, 2, and 3 subclone classes identified in this study were found to express high levels of the mesenchymal stem cells markers, CD146 and Stro1. HDF3 subclones were also associated with the strongest ALP staining detected, and strongly expressed osteoblast and cementoblast markers, including COL-I, COL-III, bone sialoprotein (BSP), and Runx2. In contrast, HDF1 subclone analyzed strongly expressed COL-I and COL-III, yet weakly expressed BSP and Runx2. The HDF2 subclone was associated with the strongest proliferative capacity. To evaluate differentiation characteristics in vivo, these various cell populations were combined with ceramic bovine bone and implanted into subcutaneous pockets of nude mice. The 30th passage of subclone HDF1 and 3 were observed to contribute to fiber collagens and the mineralized matrix present, respectively, whereas HDF2 subclones were found to have a minimal role in these formations. The formation of a cementum-periodontal ligament (PDL) complex was observed 6 weeks after HIM-DFCs (passage #30) were implanted in vivo, thus suggesting that these cells maintain stem cell characteristics. Therefore, subclone HDF1-3 may be related to the differentiation of fibroblasts in the PDL, undifferentiated cells, and osteoblasts and cementoblasts, respectively. Overall, this study is the first to amplify HIM-DFCs and associated subclones with the goal of reconstructing complex or single periodontium. Moreover, our results demonstrate the potential for this treatment approach to address periodontal defects that result from periodontitis, or for the regeneration of teeth.

Microenvironment cues received by haematopoietic stem cells (HSC) are important in regulating the choice between self-renewal and differentiation. On the basis of the differential expression of cell-surface markers, here we identify a mesenchymal stromal progenitor hierarchy, where CD45-Ter119-CD31-CD166-CD146-Sca1+(Sca1+) progenitors give rise to CD45-Ter119-CD31-CD166-CD146+(CD146+) intermediate and CD45-Ter119-CD31-CD166+CD146-(CD166+) mature osteo-progenitors. All three progenitors preserve HSC long-term multi-lineage reconstitution capability in vitro; however, their in vivo fates are different. Post-transplantation, CD146+ and CD166+ progenitors form bone only. While Sca1+ progenitors produce CD146+, CD166+ progenitors, osteocytes and CXCL12-producing stromal cells. Only Sca1+ progenitors are capable of homing back to the marrow post-intravenous infusion. Ablation of Sca1+ progenitors results in a decrease of all three progenitor populations as well as haematopoietic stem/progenitor cells. Moreover, suppressing production of KIT-ligand in Sca1+ progenitors inhibits their ability to support HSCs. Our results indicate that Sca1+ progenitors, through the generation of both osteogenic and stromal cells, provide a supportive environment for hematopoiesis.