Human malignant melanoma is a common primary malignant cutaneous tumour derived from transformed epidermal melanocytes. Patients with melanoma have a high rate of mortality due to resistance to chemotherapeutic drugs, a major obstacle to a successful treatment. Several reports have suggested that CD146 plays an important role as a signalling molecule in human melanoma. This role includes CD146 as a participant in inflammation, differentiation, adhesion, tumourigenicity, metastasis, invasion and angiogenesis among other processes, which suggests that this molecule promotes the progression of human melanoma as a multifaceted regulator. In this article, we explore the effects and corresponding mechanisms with respect to the role of CD146/MUC18 in the promotion of human melanoma progression. Collectively, the studies indicated that targeting CD146, because it is a suitable marker of poor patient outcome, might be useful in the design of future strategies for the prevention and treatment of human melanoma.

BACKGROUND

We previously reported the presence of mesenchymal stem/progenitor cells (MSCs) in inflamed pulp tissue. Here we asked whether MSCs also exist in inflamed periapical tissues resulting from endodontic infection. The objectives of this study were to detect the expression of MSC markers in periapical inflammatory tissues and to characterize isolated cells from these tissues.

METHODS

Human periapical inflammatory tissues were collected and processed to detect MSC marker expression by immunohistochemistry. Cells were isolated and tested for cell surface marker expression by using flow cytometry and examined for multiple differentiation potential into osteogenic and adipogenic pathways. In vivo formation of mineralized tissues was assessed in a mouse model.

RESULTS

Immunohistochemistry showed positive staining for MSC markers STRO-1, CD90, and CD146. Isolated cells at passage 0 appeared as typical fibroblastic cells, and a few cells formed colony-forming unit-fibroblasts (CFU-Fs). After passaging, the CFU-F forming ability diminished dramatically, and the population doubling was up to 26. Flow cytometry data showed that these cells at passage 2 expressed low levels of STRO-1 and CD146 and moderate to high levels of CD90, CD73, and CD105. At passage 6, the levels of these markers decreased. When incubated in specific differentiation medium, cells demonstrated a strong osteogenic but weak adipogenic capacity. After in vivo cell transplantation, mineralized tissues formed in immunocompromised mice.

CONCLUSIONS

Human periapical inflammatory tissues expressed MSC markers, suggesting the presence of MSCs. Isolated cells exhibited typical mesenchymal cell immunophenotype with a capacity to form mineralized matrix in vitro and in vivo.

OBJECTIVE

Endothelial cell activation and dysfunction are involved in the pathophysiology of ARDS. Circulating endothelial cells (CECs) may be a useful marker of endothelial dysfunction and damage but have been poorly studied in ARDS. We hypothesized that the CEC count may be elevated in patients with sepsis-related ARDS compared to those with sepsis without ARDS.

METHODS

ARDS was defined according to the Berlin consensus definition. The study population included 17 patients with moderate or severe ARDS, 9 with mild ARDS, 13 with sepsis and no ARDS, 13 non-septic patients, and 12 healthy volunteers. Demographic, hemodynamic, and prognostic variables, including PaO(2)/FiO(2) ratio, 28-day survival, blood lactate, APACHE II, and SOFA score, were recorded. CECs were counted in arterial blood samples using the reference CD146 antibody-based immunomagnetic isolation and UEA1-FITC staining method. Measurements were performed 12-24 h after diagnosis of ARDS and repeated daily for 3 days.

RESULTS

The median day-1 CEC count was significantly higher in patients with moderate or severe ARDS than in mild ARDS or septic-control patients [27.2 (18.3-49.4) vs. 17.4 (11-24.5) cells/ml (p < 0.034), and 18.4 (9.1-31) cells/ml (p < 0.035), respectively]. All septic patients (with or without ARDS) had higher day-1 CEC counts than the non-septic patients [19.6 (14.2-30.6) vs. 10.8 (5.7-13.2) cells/ml, p = 0.002].

CONCLUSIONS

The day-1 CEC count was significantly higher in ARDS patients than in other critically ill patients, and in moderate or severe ARDS patients compared to those with milder disease, making it a potentially useful marker of ARDS severity.

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51(+)/CD140α(+), 10.6% were CD271(+), and 0.3% were STRO-1(+)/CD146(+). Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271(+) DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271(+) DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated β-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs.

CONCLUSIONS

One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required. However, long-term growth inevitably induces cellular senescence, which potentially causes poor clinical outcomes by inducing growth arrest and the loss of stem cell properties. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This study provides strong evidence that CD146 is a novel and useful marker for predicting senescence in human umbilical cord blood-derived MSCs (hUCB-MSCs), and CD146 can potentially be applied in quality-control assessments of hUCB-MSC-based therapy.

Stro-1 has proved an efficacious marker for enrichment of skeletal stem and progenitor cells although isolated populations remain heterogeneous, exhibiting variable colony-forming efficiency and osteogenic differentiation potential. The emerging findings that skeletal stem cells originate from adventitial reticular cells have brought two further markers to the fore including CD146 and CD105 (both primarily endothelial and perivascular). This study has compared CD146-, CD105- and Stro-1 (individual and in combination)-enriched human bone marrow stromal cell subsets and assessed whether these endothelial/perivascular markers offer further selection over conventional Stro-1. Fluorescent cell sorting quantification showed that CD146 and CD105 both targeted smaller (2.22% ± 0.59% and 6.94% ± 1.34%, respectively) and potentially different human bone marrow stromal cell fractions compared to Stro-1 (16.29% ± 0.78%). CD146+, but not CD105+, cells exhibited similar alkaline phosphatase-positive colony-forming efficiency in vitro and collagen/proteoglycan deposition in vivo to Stro-1+ cells. Molecular analysis of a number of select osteogenic and potential osteo-predictive genes including ALP, CADM1, CLEC3B, DCN, LOXL4, OPN, POSTN and SATB2 showed Stro-1+ and CD146+ populations possessed similar expression profiles. A discrete human bone marrow stromal cell fraction (2.04% ± 0.41%) exhibited positive immuno-labelling for both Stro-1 and CD146. The data presented here show that CD146+ populations are comparable but not superior to Stro-1+ populations. However, this study demonstrates the critical need for new candidate markers with which to isolate homogeneous skeletal stem cell populations or skeletal stem cell populations which exhibit homogeneous in vitro/in vivo characteristics, for implementation within tissue engineering and regenerative medicine strategies.

Muscle-derived cells have been successfully isolated using a variety of different methods and have been shown to possess multilineage differentiation capacities, including an ability to differentiate into articular cartilage and bone in vivo; however, the characterization of human muscle-derived stem cells (hMDSCs) and their bone regenerative capacities have not been fully investigated. Genetic modification of these cells may enhance their osteogenic capacity, which could potentially be applied to bone regenerative therapies. We found that hMDSCs, isolated by the preplate technique, consistently expressed the myogenic marker CD56, the pericyte/endothelial cell marker CD146, and the mesenchymal stem cell markers CD73, CD90, CD105, and CD44 but did not express the hematopoietic stem cell marker CD45, and they could undergo osteogenic, chondrogenic, adipogenic, and myogenic differentiation in vitro. In order to investigate the osteoinductive potential of hMDSCs, we constructed a retroviral vector expressing BMP4 and GFP and a lentiviral vector expressing BMP2. The BMP4-expressing hMDSCs were able to undergo osteogenic differentiation in vitro and exhibited enhanced mineralization compared to nontransduced cells; however, when transplanted into a calvarial defect, they failed to regenerate bone. Local administration of BMP4 protein and cell pretreatment with N-acetylcysteine (NAC), which improves cell survival, did not enhance the osteogenic capacity of the retro-BMP4-transduced cells. In contrast, lenti-BMP2-transduced hMDSCs not only exhibited enhanced in vitro osteogenic differentiation but also induced robust bone formation and nearly completely healed a critical-sized calvarial defect in CD-1 nude mice 6 weeks following transplantation. Herovici's staining of the regenerated bone demonstrated that the bone matrix contained a large amount of type I collagen. Our findings indicated that the hMDSCs are likely mesenchymal stem cells of muscle origin and that BMP2 is more efficient than BMP4 in promoting the bone regenerative capacity of the hMDSCs in vivo.

Bone tissue has a significant potential for healing, which involves a significant the interplay between bone and immune cells. While fracture healing represents a useful model to investigate endochondral bone healing, intramembranous bone healing models are yet to be developed and characterized. In this study, a micro-computed tomography, histomorphometric and molecular (RealTimePCRarray) characterization of post tooth-extraction alveolar bone healing was performed on C57Bl/6 WT mice. After the initial clot dominance (0 h), the development of a provisional immature granulation tissue is evident (7 d), characterized by marked cell proliferation, angiogenesis and inflammatory cells infiltration; associated with peaks of growth factors (BMP-2-4-7,TGFβ1,VEGFa), cytokines (TNFα, IL-10), chemokines & receptors (CXCL12, CCL25, CCR5, CXCR4), matrix (Col1a1-2, ITGA4, VTN, MMP1a) and MSCs (CD105, CD106, OCT4, NANOG, CD34, CD146) markers expression. Granulation tissue is sequentially replaced by more mature connective tissue (14 d), characterized by inflammatory infiltrate reduction along the increased bone formation, marked expression of matrix remodeling enzymes (MMP-2-9), bone formation/maturation (RUNX2, ALP, DMP1, PHEX, SOST) markers, and chemokines & receptors associated with healing (CCL2, CCL17, CCR2). No evidences of cartilage cells or tissue were observed, strengthening the intramembranous nature of bone healing. Bone microarchitecture analysis supports the evolving healing, with total tissue and bone volumes as trabecular number and thickness showing a progressive increase over time. The extraction socket healing process is considered complete (21 d) when the dental socket is filled by trabeculae bone with well-defined medullary canals; it being the expression of mature bone markers prevalent at this period. Our data confirms the intramembranous bone healing nature of the model used, revealing parallels between the gene expression profile and the histomorphometric events and the potential participation of MCSs and immune cells in the healing process, supporting the forthcoming application of the model for the better understanding of the bone healing process.

Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk.

BACKGROUND

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Recurrence and metastasis after curative resection remain critical obstacles in HCC treatment. CD146 predicted poor prognosis of a variety of cancers including melanoma, breast tumors, prostate cancer, and gastric cancer. However, the role of CD146 in HCC has not yet been systematically explored.

METHODS

To investigate the role of CD146 in HCC, we evaluated its expression in HCC tissues and HCC cell lines using real-time PCR and western blotting (WB). Second, we established HCC cell lines that stably overexpressed and interfered CD146 and explored the function of CD146 in HCC in vitro and in vivo. Third, we conducted microarray analysis to investigate the potential mechanism by identifying differentially expressed genes. Last, follow ups were conducted to help uncover the connection of CD146 expression and the prognosis of HCC patients.

RESULTS

We found that CD146 was overexpressed in HCC tissues and that high CD146 expression predicted poor overall survival time and shorter recurrence period in HCC patients. In vitro and in vivo experiments indicated that CD146 promoted migration and invasion of HCC cell lines. Further study indicated that CD146 promoted epithelial mesenchymal transition (EMT), IL-8 upregulation, and STAT1 downregulation. CD146 was upregulated in HCC tissues and cell lines.

CONCLUSIONS

CD146 promoted metastasis of HCC cells and predicted poor prognosis of HCC patients. CD146 induced EMT, and IL-8 upregulation and STAT1 downregulation may be the potential underlying mechanism. The exact mechanism still needs further investigation.

BACKGROUND

Circulating endothelial cells (CECs) have emerged as non-invasive biomarkers of vascular dysfunction. The most widely used method for their detection is CD146-based immunomagnetic separation (IMS). Although this approach has provided consensus values in both normal and pathologic situations, it remains tedious and requires a trained operator.

OBJECTIVE

Our objective was to evaluate a new hybrid assay for CEC measurement using a combination of pre-enrichment of CD146+ circulating cells and multiparametric flow cytometry measurement (FCM).

METHODS

CECs were determined in peripheral blood from 20 healthy volunteers, 12 patients undergoing coronary angioplasty, and 30 renal transplant recipients, and blood spiked with cultured endothelial cells. CD146+ cells were isolated using CD146-coated magnetic nanoparticles and labeled using CD45-fluorescein isothiocyanate and CD146-PE or isotype control antibody and propidium iodide before FCM. The same samples were also processed using CD146-based immunomagnetic separation as the reference method.

RESULTS

The hybrid assay detected CECs, identified as CD45(dim)/CD146(bright)/propidium iodide(+), with high size-related scatter characteristics, and clearly discriminated these from CD45(bright)/CD146(dim) activated T lymphocytes. The method demonstrated both high recovery efficiency and good reproducibility. Both IMS and the hybrid assay similarly identified increased CEC levels in patients undergoing coronary angioplasty and renal transplantation, when compared to healthy controls. In patients, CEC values from these two methods were of the same order of magnitude and highly correlated. Bland-Altman analysis revealed poor statistical agreement between methods, flerrofluid-FCM providing higher values than IMS.

CONCLUSIONS

This new hybrid FCM assay constitutes an accurate alternative to visual counting of CECs following CD146-based IMS.

BACKGROUND

Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)(low-to-intermedate), sideward light scatter (SSC)(low), CD45(-), CD31(++) and CD146(+) is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658-3661). Here, we set out to confirm the endothelial nature of these cells.

METHODS

We isolated cells with a FSC(low-to-intermediate), SSC(low), CD31(++), CD45(dim) immunophenotype (termed "cells meeting our immunophenotypic criteria for endothelial cells" [CMOIC]) from healthy donors to study the expression of endothelium-associated markers using several techniques. Special attention was paid to reagents identifying the endothelial cell-specific marker CD146. We compared antigen expression patterns of CMOIC with those of the HUVEC endothelial cell line and lymphocytes. Electron microscopy was used to detect the presence of endothelial cell-specific Weibel-Palade bodies in the sorted cells.

RESULTS

CD146 expression was negative on CMOIC for all tested CD146 mAbs, but positive on HUVEC cells and a minor subset of T lymphocytes. Using flow cytometry, we found no expression of any endothelium-associated marker except for CD31 and CD34. HUVEC cells were positive for all endothelial markers except for CD34. Evaluation of CMOIC morphology showed a homogenous population of cells with a highly irregular nucleus-like structure and positive endothelial immunohistochemistry. CMOIC contained neither nuclei nor DNA. Electron microscopy revealed the absence of a nucleus, the absence of endothelial specific Weibel-Palade bodies, and revealed CMOIC to be large platelets.

CONCLUSIONS

The vast majority of cells with the immunophenotype FSC(low-to-intermediate), SSC(low), CD45(-), CD31(++) do not express CD146 and are large platelets rather than endothelial cells.

Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS) as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs) from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total) tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC) including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.

Skeleton and liver are preferred organs for cancer dissemination in metastatic melanoma negatively impacting quality of life, therapeutic success and overall survival rates. At the target organ, the local microenvironment and cell-to-cell interactions between invading and resident stromal cells constitute critical components during the establishment and progression of metastasis. Mesenchymal stem cells (MSCs) possess, in addition to their cell progenitor function, a secretory capacity based on cooperativity with other cell types in injury sites including primary tumors (PT). However, their role at the target organ microenvironment during cancer dissemination is not known. We report that local MSCs, acting as pericytes, regulate the extravasation of melanoma cancer cells (MCC) specifically to murine bone marrow (BM) and liver. Intra-arterially injected wild-type MCC fail to invade those selective organs in a genetic model of perturbed pericyte coverage of the vasculature (PDGF-B(ret/ret)), similar to CD146-deficient MCC injected into wild type mice. Invading MCC interact with resident MSCs/pericytes at the perivascular space through co-expressed CD146 and Sdf-1/CXCL12-CXCR4 signaling. Implanted engineered bone structures with MSCs/pericytes deficient of either Sdf-1/CXCL12 or CD146 become resistant to invasion by circulating MCC. Collectively, the presence of MSCs/pericytes surrounding the target organ vasculature is required for efficient melanoma metastasis to BM and liver.

BACKGROUND

The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

RESULTS

By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7-14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

CONCLUSIONS

Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

In this report, we use zebrafish as a model system to understand the importance of CD146 in vascular development. Endothelial-specific expression of CD146 was verified by whole-mount in situ hybridization. Suppression of CD146 protein expression by antisense morpholino oligonucleotides (MO) resulted in poorly developed intersomitic vessels (ISVs). In CD146 morphants, we observed a lack of blood flow through the ISV region, despite that fluorescence microangiography showed that the ISVs were present. This finding suggests that the lumens of the developing ISVs may be too narrow for proper circulation. Additionally, remodeling of the caudal vein plexus into functional vascular tubes appeared to be affected. Suppression of CD146 protein expression resulted in a circulation shunt that caused the circulation to by-pass part of the caudal artery/vein system. The same vascular defects were recapitulated by using a second morpholino oligonucleotide. This morphant expressed a truncated CD146 protein with amino acids V32 to T57 at the N terminus deleted in an in-frame manner. This region, therefore, is likely to contain elements critical for CD146 function. This study provides the first in vivo functional assessment of CD146 in embryonic development by showing that knockdown of CD146 protein expression severely hinders vascular development in zebrafish.

Whereas the conventional tissue engineering strategy involves the use of scaffolds combined with appropriate cell types to restore normal functions, the concept of in situ tissue regeneration uses host responses to a target-specific scaffold to mobilize host cells to a site of injury without the need for cell seeding. For this purpose, local delivery of bioactive molecules from scaffolds has been generally used. However, this approach has limited stem cell recruitment into the implants. Thus, we developed a combination of systemic delivery of substance P (SP) and local release of stromal-derived factor-1α (SDF-1α) from an implant. In this study, we examined whether this combined system would significantly enhance recruitment of host stem cells into the implants. Flow cytometry and immunohistochemistry for CD29/CD45, CD146/α-smooth muscle actin, and c-kit demonstrated that this system significantly increased the number of stem cell-like cells within the implants when compared with other systems. In vitro culture of the cells that had infiltrated into the scaffolds from the combined system confirmed that host stem cells were recruited into these implants and indicated that they were capable of differentiation into multiple lineages. These results indicate that this combined system may lead to more efficient tissue regeneration.

Marrow stromal cells constitute a heterogeneous population of cells, typically isolated after expansion in culture. In vivo, stromal cells often exist in close proximity or in direct contact with monocyte-derived macrophages, yet their interaction with monocytes is largely unexplored. In this report, isolated CD146(+) and CD146(-) stromal cells, as well as immortalized cell lines representative of each (designated HS27a and HS5, respectively), were shown by global DNase I hypersensitive site mapping and principal coordinate analysis to have a lineage association with marrow fibroblasts. Gene expression profiles generated for the CD146(+) and CD146(-) cell lines indicate significant differences in their respective transcriptomes, which translates into differences in secreted factors. Consequently, the conditioned media (CM) from these two populations induce different fates in peripheral blood monocytes. Monocytes incubated in CD146(+) CM acquire a tissue macrophage phenotype, whereas monocytes incubated in CM from CD146(-) cells express markers associated with pre-dendritic cells. Importantly, when CD14(+) monocytes are cultured in contact with the CD146(+) cells, the combined cell populations, assayed as a unit, show increased levels of transcripts associated with organismal development and hematopoietic regulation. In contrast, the gene expression profile from cocultures of monocytes and CD146(-) cells does not differ from that obtained when monocytes are cultured with CD146(-) CM. These in vitro results show that the CD146(+) marrow stromal cells together with monocytes increase the expression of genes relevant to hematopoietic regulation. In vivo relevance of these data is suggested by immunohistochemistry of marrow biopsies showing juxtaposed CD146(+) cells and CD68(+) cells associated with these upregulated proteins.

We demonstrate that subpopulations of adult human skeletal muscle-derived stem cells, myogenic endothelial cells (MECs), and perivascular stem cells (PSCs) can be simultaneously purified by fluorescence-activated cell sorting (FACS) from cryopreserved human primary skeletal muscle cell cultures (cryo-hPSMCs). For FACS isolation, we utilized a combination of cell lineage markers: the myogenic cell marker CD56, the endothelial cell marker UEA-1 receptor (UEA-1R), and the perivascular cell marker CD146. MECs expressing all three cell lineage markers (CD56(+)UEA-1R(+)CD146(+)/CD45(-)) and PSCs expressing only CD146 (CD146(+)/CD45(-)CD56(-)UEA-1R(-)) were isolated by FACS. To evaluate their myogenic capacities, the sorted cells, with and without expansion in culture, were transplanted into the cardiotoxin-injured skeletal muscles of immunodeficient mice. The purified MECs exhibited the highest regenerative capacity in the injured mouse muscles among all cell fractions tested, while PSCs remained superior to myoblasts and the unpurified primary skeletal muscle cells. Our findings show that both MECs and PSCs retain their high myogenic potentials after in vitro expansion, cryopreservation, and FACS sorting. The current study demonstrates that myogenic stem cells are prospectively isolatable from long-term cryopreserved primary skeletal muscle cell cultures. We emphasize the potential application of this new approach to extract therapeutic stem cells from human muscle cells cryogenically banked for clinical purposes.

In recent years several studies have been supporting the existence of a close relationship in terms of function and progeny between Mesenchymal Stem Cells (MSCs) and Pericytes. This concept has opened new perspectives for the application of MSCs in Tissue Engineering (TE), with special interest for the pre-vascularization of cell dense constructs. In this work, cell sheet technology was used to create a scaffold-free construct composed of osteogenic, endothelial and perivascular-like (CD146(+)) cells for improved in vivo vessel formation, maturation and stability. The CD146 pericyte-associated phenotype was induced from human bone marrow mesenchymal stem cells (hBMSCs) by the supplementation of standard culture medium with TGF-β1. Co-cultured cell sheets were obtained by culturing perivascular-like (CD146(+)) cells and human umbilical vein endothelial cells (HUVECs) on an hBMSCs monolayer maintained in osteogenic medium for 7 days. The perivascular-like (CD146(+)) cells and the HUVECs migrated and organized over the collagen-rich osteogenic cell sheet, suggesting the existence of cross-talk involving the co-cultured cell types. Furthermore the presence of that particular ECM produced by the osteoblastic cells was shown to be the key regulator for the singular observed organization. The osteogenic and angiogenic character of the proposed constructs was assessed in vivo. Immunohistochemistry analysis of the explants revealed the integration of HUVECs with the host vasculature as well as the osteogenic potential of the created construct, by the expression of osteocalcin. Additionally, the analysis of the diameter of human CD146 positive blood vessels showed a higher mean vessel diameter for the co-cultured cell sheet condition, reinforcing the advantage of the proposed model regarding blood vessels maturation and stability and for the in vitro pre-vascularization of TE constructs.

BACKGROUND

In a phase 1 study, we investigated whether interferon beta reduced endothelial damage in patients with cardiac persistence of human parvovirus B19 (B19V) infection.

RESULTS

In vitro, B19V infected cultivated endothelial cells (ECs), which led to a reduction in their viability (P = .007). Interferon beta suppressed B19V replication by 63% (P = .008) in ECs and increased their viability (P = .021). Circulating mature apoptotic ECs (CMAECs [CD45(-)CD146(+) cells expressing von Willebrand factor and annexin V]) and circulating progenitor cells (CPCs [CD34(+)KDR(+) cells]) were quantified by flow cytometry in 9 symptomatic patients with cardiac B19V infection before and after 6 months of interferon beta therapy (16 MU) and were compared to levels in 9 healthy control subjects. Endothelial dysfunction was measured using flow-mediated dilatation of the forearm. Patients with B19V persistence had significantly higher (P = .04) levels of CMAECs than did control subjects, which normalized after treatment (mean +/- standard deviation, 0.06% +/-0.08% vs 0.01% +/- 0.006%; P = .008). Similar improvement was shown for flow-mediated dilatation (P = .04) in the treatment group only (P = .017 for the comparison with untreated patients with B19V persistence n = 5). There were significantly higher numbers of CPCs in patients with B19V persistence before therapy (mean +/- standard deviation, 0.04% +/- 0.05% vs 0.01% +/- 0.004%; P = .02; than in control subjects, which normalized after treatment (P = .03).

CONCLUSIONS

Thus, we present (for the first time, to our knowledge) a modulation of virally induced chronic endothelial damage-specifically, EC apoptosis and endothelial regeneration.

The cell surface glycoprotein MUC18MCAM/CD146 was originally defined as a marker of melanoma progression and has been suspected to be directly linked to the metastatic process of this malignancy. In order to address this question, 2 MCAM negative human melanoma cell lines, SK-2 and XP44RO(Mel), were transfected with MCAM-encoding cDNA. Surface MCAM expression on SK-2 and XP44RO(Mel) transfectants was similar to that observed in naturally occurring MCAM positive human melanoma cells and transfectants demonstrated MCAM-dependent increase in homotypic adhesion in vitro. The growth behavior of 7 MCAM transfectants and their respective vector controls was evaluated in SCID mice. Tumor size at 4-5 weeks after s.c. implantation was highly variable, but did not correlate with MCAM expression. Despite massive primary tumor formation at the injection site, no spontaneous metastasis was observed with any of the investigated MCAM transfectants. The influence of MCAM expression on lung metastases formation in an experimental metastasis assay was system dependent, converting only XP44RO(Mel) transfectants into metastatic cells, although increased homotypic adhesion, leading to formation of tumor cell clusters, was observed with transfectants of both cell lines in vitro. Our findings indicate that MCAM expression of human melanoma cells has an influence on later stages of the metastatic process only, namely, extravasation and establishment of new foci of growth, but is per se not sufficient for this process.

It has been shown that injecting a suspension of IFN-γ-secreting tumor cells results in their rejection. This effect has been attributed to IFN-γ preventing tumor stroma formation but not to a direct effect on the cancer cells. However, it is not known, which influence IFN-γ has on tumors with an established stroma. To address this question, the plasmacytoma cell line J558L was transduced with a vector allowing doxycycline-inducible IFN-γ gene expression. After the injection of the tumor cells into mice, IFN-γ was induced at different time points. Tumors did not grow when inducing IFN-γ immediately after tumor cell inoculation, while approximately half of the tumors were rejected when IFN-γ was induced in early established tumors within 2 weeks. Induction of IFN-γ 2-3 weeks after tumor cell inoculation was less efficient (0-17% rejection). IFN-γ induction in established tumors led to a reduction of CD146(+) endothelial cells and massive necrosis. Together, we show that vascularized tumors can be rejected by local IFN-γ expression, but that rejection of established tumors was less efficient over time. This suggests that transplanted tumors became less susceptible to local IFN-γ treatment the better they are established.

Human mesenchymal stromal cells derived from bone marrow (BMSC) and adipose tissue (ATSC) represent a valuable source of progenitor cells for cell therapy and tissue engineering. While ectopic bone formation is a standard activity of human BMSC on calcium phosphate ceramics, the bone formation capacity of human ATSC has so far been unclear. The objectives of this study were to assess the therapeutic potency of ATSC for bone formation in an ectopic mouse model and determine molecular differences by standardized comparison with BMSC. Although ATSC contained less CD146(+) cells, exhibited better proliferation and displayed similar alkaline phosphatase activity upon osteogenic in vitro differentiation, cells did not develop into bone-depositing osteoblasts on β-TCP after 8weeks in vivo. Additionally, ATSC expressed less BMP-2, BMP-4, VEGF, angiopoietin and IL-6 and more adiponectin mRNA, altogether suggesting insufficient osteochondral commitment and reduced proangiogenic activity. Chondrogenic pre-induction of ATSC/β-TCP constructs with TGF-β and BMP-6 initiated ectopic bone formation in >75% of samples. Both chondrogenic pre-induction and the osteoconductive microenvironment of β-TCP were necessary for ectopic bone formation by ATSC pointing towards a need for inductive conditions/biomaterials to make this more easily accessible cell source attractive for future applications in bone regeneration.