Dental pulp stem cells were primarily derived from the pulp tissues of exfoliated deciduous teeth, primary incisors and permanent third molar teeth. The aim of this study was to isolate and extensively characterise SCs derived from human natal dental pulp (hNDP). For characterisation, proliferation capacity, phenotypic properties, ultrastructural and differentiation characteristics and gene expression profiles were utilised. A comparison was done between the properties of NDP-SCs and the properties of mesenchymal stem cells (MSCs) from bone marrow (BM) of the human. Stem cells isolated from hNDP and hBM were analysed by flow cytometry, reverse transcriptase-PCR, Real Time-PCR, and immunocytochemistry. Both cell lines were directionally differentiated towards adipogenic, osteogenic chondrogenic, myogenic and neurogenic lineages. hNDP-SCs and hBM-MSCs expressed CD13, CD44, CD90, CD146 and CD166, but not CD3, CD8, CD11b, CD14, CD15, CD19, CD33, CD34, CD45, CD117, and HLA-DR. Ultrastructural characteristics of hNDP-SCs showed more developed and metabolically active cells. hNDP-SCs and hBM-MSCs expressed some adipogenic (leptin, adipophilin and PPARgamma), myogenic (desmin, myogenin, myosinIIa, and alpha-SMA), neurogenic (gamma-enolase, MAP2a,b, c-fos, nestin, NF-H, NF-L, GFAP and betaIII tubulin), osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, and type I collagen) and chondrogenic (type II collagen, SOX9) markers without any stimulation towards differentiation under basal conditions. Embryonic stem cell markers Oct4, Rex-1, FoxD-3, Sox2, and Nanog were also identified. The differentiation potential of hNDP-SCs and hBM-MSCs to adipogenic, osteogenic, chondrogenic, myogenic and neurogenic was shown. This report described the first successful isolation and characterisation of hNDP-SCs.

OBJECTIVE

Circulating endothelial cells (CECs) and progenitor cells are currently evaluated as potential biomarkers of antiangiogenic therapy. CD146 is considered a panendothelial-specific marker, but its utility as a CEC marker in cancer patients remains unclear.

METHODS

We analyzed the expression of CD146 on mononuclear blood cells, primary tissue endothelial cells, and malignant and normal tissues by flow cytometric and immunohistochemical analyses. Furthermore, we measured the circulation kinetics of CD146+ cells before, and then 3 and 12 days after vascular endothelial growth factor (VEGF) antibody blockade by bevacizumab infusion in rectal cancer patients enrolled in a phase I trial.

RESULTS

In the peripheral blood of these cancer patients, over 90% of the CD146+ cells were CD45+ hematopoietic cells. CD146 expression was primarily detected on a subset of CD3+CD4+ lymphocytes, and was undetectable on CD34+CD133+CD45(dim) progenitor cells or CD31(bright)CD45- viable CECs. In contradistinction, CD146 was detectable in tissues on both cellular components of tumor vessel wall: CD31(bright)CD45- endothelial cells and alpha-SMA+ pericytes. Unlike viable CECs and progenitor cells, CD146+ cell concentration in the peripheral blood of cancer patients did not decrease during VEGF blockade.

CONCLUSIONS

CD146 is fairly homogeneously expressed on vascular endothelium but not on viable CECs or progenitor cells. The vast majority of CD146+ blood cells are lymphocytes, and VEGF blockade by bevacizumab did not significantly change their number in rectal cancer patients. These results underscore the need for further characterization and identification of new markers for CEC subpopulations for their development as biomarkers of antiangiogenic therapy.

We previously reported a unique CD14(+)CD45(+)CD34(+) type I collagen(+) cell fraction derived from human circulating CD14(+) monocytes, named monocyte-derived multipotential cells (MOMCs). This primitive cell population contains progenitors capable of differentiating along the mesenchymal and neuronal lineages. Here, we investigated whether MOMCs can also differentiate along the endothelial lineage. MOMCs treated with angiogenic growth factors for 7 days changed morphologically and adopted a caudate appearance with rod-shaped microtubulated structures resembling Weibel-Palade bodies. Almost every cell expressed CD31, CD144, vascular endothelial growth factor (VEGF) type 1 and 2 receptors, Tie-2, von Willebrand factor (vWF), endothelial nitric-oxide synthase, and CD146, but CD14/CD45 expression was markedly downregulated. Under these culture conditions, the MOMCs continued to proliferate for up to 7 days. Functional characteristics, including vWF release upon histamine stimulation and upregulated expression of VEGF and VEGF type 1 receptor in response to hypoxia, were indistinguishable between the MOMC-derived endothelial-like cells and cultured mature endothelial cells. The MOMCs responded to angiogenic stimuli and promoted the formation of mature endothelial cell tubules in Matrigel cultures. Finally, in xenogenic transplantation studies using a severe combined immunodeficient mouse model, syngeneic colon carcinoma cells were injected subcutaneously with or without human MOMCs. Cotransplantation of the MOMCs promoted the formation of blood vessels, and more than 40% of the tumor vessel sections incorporated human endothelial cells derived from MOMCs. These findings indicate that human MOMCs can proliferate and differentiate along the endothelial lineage in a specific permissive environment and thus represent an autologous transplantable cell source for therapeutic neovasculogenesis.

BACKGROUND

Bone marrow derived mesenchymal stem cells (BM-MSCs) are used extensively in transplantation but their use is associated with many problems including low abundance in BM, low overall number, decreased differentiation potential with age and the invasive isolation procedures needed to obtain BM. We report a novel method of isolating placental MSCs (pMSCs) from chorionic villi, which exhibit the phenotypic and functional characteristics that will make them an attractive source of MSCs for cell-based therapy.

METHODS

A novel explant approach was used to isolate pMSCs from chorionic villi of human placentae. These pMSCs were characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation medium as demonstrated by cytochemical staining. The gene and protein expression profiles of pMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, cytokine secretion by pMSCs was also analysed using sandwich enzyme-linked immunosorbent assay (ELISA) technique. Moreover, the migration and proliferation potentials of pMSCs were also determined.

RESULTS

pMSCs were isolated from fetal part of the chorionic villi and these pMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these pMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed several adhesion molecules, chemokines/receptors, growth factor receptors and cytokines/receptors. Moreover, they secreted many cytokines (IL-1Ra, IL6, IL8, IL10, IL11 and IL15) and they were able to proliferate. Furthermore, they migrated in response to chemotactic factors including stromal cell-derived factor-1 (SDF-1), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1).

CONCLUSIONS

We devised a novel explant method of isolating pMSCs that expressed many biological factors responsible for mediating cellular processes such as migration/homing, immune modulation and angiogenesis. Therefore, we suggest that pMSCs prepared from human term placental chorionic villous explants are an attractive source of MSCs for cell therapy.

BACKGROUND

Endothelial dysfunction is an important factor in the pathogenesis of atherosclerosis, and endothelial microparticles (EMPs) are considered as markers of endothelial dysfunction. In this study, we aimed to examine the relationship between EMPs and arterial stiffness and atherosclerosis in children with chronic kidney disease (CKD).

METHODS

This cross-sectional study included 37 dialysis patients (12 haemodialysis, 25 peritoneal dialysis), 33 pre-dialysis patients and 18 healthy controls. Both in vivo and in vitro (HUVECs) evaluations were used for the study. Circulating EMPs were measured by flow cytometry. The carotid artery intima-media thickness (cIMT) and pulse wave velocity (PWV) were measured by using high-resolution ultrasound. Study groups were compared for circulating EMP, cIMT and PWV. The relationship between EMPs and arterial stiffness and atherosclerosis was evaluated.

RESULTS

The levels of PWV, cIMT, CD144 + EMP and CD146 + EMP in the dialysis group were significantly higher than those in the pre-dialysis and control groups (P < 0.05). Additionally, the levels of cIMT, CD144 + EMP and CD146 + EMP in the pre-dialysis group were significantly higher than those in the control group (P < 0.05). In all CKD patients, the CD144 + EMP was significantly positively associated with blood pressures, age, known duration of disease, CRP and PTH, and was significantly negatively associated with haemoglobin, GFR and albumin. The CD146 + EMP was significantly positively associated with blood pressures, age and CRP. In a multiple linear regression analysis, in the CKD group, cIMT was independently related to mean blood pressure and dialysis duration. PWV was independently related to the CD144 + EMP and mean blood pressure.

CONCLUSIONS

Our results suggest that endothelial damage starts in the early stage of CKD, that the endothelial dysfunction becomes overt with the increase of cardiovascular risk factors and that EMPs may be a reliable marker of the subclinical atherosclerosis and arterial stiffness.

OBJECTIVE

Endothelial cells play a central pathogenetic role in ANCA-associated small-vessel vasculitis (AAV). Circulating endothelial cells (CECs), as a marker of endothelial damage, have been shown to be elevated in vasculitis. More recently, endothelial microparticles (EMPs) were found to be increased in active childhood vasculitis. The role of EMP in adult AAV and the relationship between EMP and CEC is unclear.

METHODS

We studied 26 patients with AAV, 12 healthy volunteers and 10 patients with IgA nephropathy as disease control. Platelet-poor plasma was ultracentrifuged. MPs were identified and enumerated with flow cytometry, Annexin V, CD62E and CD105 antibodies. Leucocyte- and platelet-derived MPs were also measured. CEC were isolated and enumerated with CD146-driven immuno-magnetic isolation.

RESULTS

EMPs are significantly elevated in patients with active vasculitis (CD62E: mean 248 MP/microl +/- 198 s.d.; CD105: 121 +/- 135/microl) compared with patients in remission/partial remission (CD62E: 55 +/- 30/microl, P = 0.001; CD105: 16 +/- 12/microl, P = 0.002) and healthy volunteers (CD62E: 66 +/- 33/microl, P = 0.002; CD105: 25 +/- 26/microl, P = 0.007). The MP count correlates with disease activity measured by the Birmingham Vasculitis Activity Score (BVAS) (CD62E: r = 0.703; CD105: r = 0.445, P < 0.023).

CONCLUSIONS

EMPs are elevated in active adult AAV. EMP levels correlate with disease activity and might serve as a marker of endothelial activation and damage. Differential detection of endothelial, platelet- and leucocyte-derived MPs may provide more insight in to the pathogenesis of AAV.

Endometrial cell clones derived from in vitro cultured purified stromal cells obtained by endometrial biopsy display characteristic stem cell features, including clonality; long-term culturing properties; multilineage differentiation potential; expression of CD146, CD105, CD90, CD73, MSI1, NOTCH1, and SOX2; and absence of CD34 and CD14 expression. We conclude that adult stem cells are present in endometrial biopsies performed in a routine clinical setting, offering new large-scale approaches for future research, diagnostics, and therapies involving adult stem cells.

Bone marrow mesenchymal stem cells (MSCs) are plastic adherent cells that can differentiate into various tissue lineages, including osteoblasts, adipocytes and chondrocytes. However, this progenitor property is not shared by all cells within the MSC population. In addition, MSCs vary in their proliferation capacity and expression of markers. Because of heterogeneity of CD146 expression in the MSC population, we compared CD146(-/Low) and CD146(High) cells under clonal conditions and after sorting of the non-clonal cell population to determine whether this expression is associated with specific functions. CD146(-/Low) and CD146(High) bone marrow MSCs did not differ in colony-forming unit-fibroblast number, osteogenic, adipogenic and chondrogenic differentiation or in vitro haematopoietic-supportive activity. However, CD146(-/Low) clones proliferated slightly but significantly faster than did CD146(High) clones. In addition, a strong expression of CD146 molecule was associated with a commitment to a vascular smooth muscle cell (VSMC) lineage characterized by a strong up-regulation of calponin-1 and SM22α expression and an ability to contract collagen matrix. Thus, within a bone marrow MSC population, certain subpopulations characterized by high expression of CD146, are committed towards a VSMC lineage.

Circulating endothelial cells (CECs) were first described over 30 years ago in smears of peripheral blood. Since then, more sophisticated techniques for CEC isolation have become available. In particular, immunomagnetic isolation and fluorescence-activated cell sorting (FACS) have been employed with success. We provide a short historical perspective and a comprehensive review on the subject. We review isolation and enumeration of CECs with an emphasis on CD146-driven immunomagnetic isolation and FACS. We describe, in great detail, advantages and pitfalls of both approaches and compare their specificity. Moreover, we provide a comprehensive list of clinical studies in this field and describe the possible clinical use of CECs. We also describe the phenotype of these cells and list typical surface markers. In addition, we review the phenotype of CECs and discuss mechanisms of detachment. We speculate about potential interactions between CECs and other cell subsets. We also describe other serum markers of endothelial damage and compare CECs with these markers. Finally, we highlight differences between circulating endothelial cells and endothelial progenitor cells. In summary, CECs must now be regarded as a sensitive and specific marker of endothelial damage. We emphasize that use of CECs in a clinical setting is on the horizon and pathogenetic clues may also be obtained.

BACKGROUND

The influence of COPD exacerbation on the endothelium is not completely understood. Circulating endothelial microparticles (EMPs) are membrane vesicles in circulating blood that are shed by activated or apoptotic endothelial cells.

OBJECTIVE

To compare EMP numbers in stable COPD patients with those during and after exacerbation.

METHODS

We examined the EMP numbers in 80 stable COPD patients, 27 patients with exacerbated COPD, and 20 healthy non-COPD volunteers. EMPs were defined as CD144+ MPs (VE-cadherin EMPs), CD31+/CD41- MPs (PECAM EMPs), CD146 MPs (MCAM EMPs) and CD62E+ EMPs (E-selectin EMPs) as analysed by FACS. Von Willebrand factor (vWF) expression was utilised to identify the origins of the EMPs.

RESULTS

VE-cadherin, PECAM and E-selectin EMP numbers were significantly higher in the stable COPD patients than in the non-COPD volunteers, and they were significantly higher in the patients with exacerbated COPD than in the stable COPD patients. The majority of these increased EMPs were vWF-negative, indicating a pulmonary capillary origin. Baseline E-selectin EMP levels were significantly higher in COPD patients who experienced frequent exacerbations than in those who did not have frequent exacerbations (p<0.001). Twenty-eight days after the onset of exacerbation, E-selectin EMP levels returned to those observed in stable COPD patients, whereas PECAM EMP levels remained high. MCAM EMP numbers were not elevated in stable or exacerbated-COPD patients.

CONCLUSIONS

Endothelial damage, mainly in pulmonary capillaries, occurs during exacerbation and continues even after clinical symptoms disappear. Higher baseline E-selectin EMP levels may indicate COPD patients who are susceptible to exacerbation.

Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, "normal-like" cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [J Natl Cancer Inst 101(1):61-66, 2009]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients.

Perivascular multipotent mesenchymal progenitors exist in a variety of tissues, including the placenta. Here, we suggest that the abundant vasculature present in the human placenta can serve as a source of myogenic cells to regenerate skeletal muscle. Chorionic villi dissected from the mid-gestation human placenta were first transplanted intact into the gastrocnemius muscles of SCID/mdx mice, where they participated in muscle regeneration by producing myofibers expressing human dystrophin and spectrin. In vitro-cultured placental villi released rapidly adhering and migratory CD146+CD34⁻CD45⁻CD56⁻ cells of putative perivascular origin that expressed mesenchymal stem cell markers. CD146+CD34⁻CD45⁻CD56⁻ perivascular cells isolated and purified from the placental villi by flow cytometry were indeed highly myogenic in culture, and generated dystrophin-positive myofibers, and they promoted angiogenesis after transplantation into SCID/mdx mouse muscles. These observations confirm the existence of mesenchymal progenitor cells within the walls of human blood vessels, and suggest that the richly vascularized human placenta is an abundant source of perivascular myogenic cells able to migrate within dystrophic muscle and regenerate myofibers.

Superparamagnetic iron oxide nanoparticles (SPION) are increasingly used to label human bone marrow stromal cells (BMSCs, also called "mesenchymal stem cells") to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB) staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the "stemness" of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the "gold standard" of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs.

The chorionic villi of human term placentae are a rich source of mesenchymal stem cells (PMSCs). The stem cell "niche" within the chorionic villi regulates how PMSCs participate in placental tissue generation, maintenance and repair, but the anatomic location of the niche has not been defined. A number of cell surface markers for phenotypic characterisation of mesenchymal stem cells (MSCs) were employed to identify the stem cell niche within the chorionic villi of first trimester and term human placenta. This included antibodies to pericyte cell surface markers STRO-1 and 3G5, which have been used to identify mesenchymal stem cells in other tissues, but have not been studied in placental tissues. PMSCs were isolated from term human placentae and shown to have stem cell properties by their ability to grow on untreated plastic culture ware, capacity for forming clones (i.e. clonogenicity) and their capability to differentiate into adipocytes, chondrocytes and osteocytes. Western analysis confirmed that STRO-1 and 3G5 are present in placental protein extracts and in PMSCs. Immunocytochemistry revealed PMSCs were positive for MSC cell surface markers (STRO-1, 3G5, CD105, CD106, CD146, CD49a, alpha-SMA) and negative for haematopoietic stem cell markers (CD117, CD34) and endothelial markers (CD34, vWF). Immunohistochemistry with antibodies to MSC cell surface markers on first trimester and term tissues revealed a vascular niche for PMSCs. Dual-label immunofluorescence analysis was used to compare STRO-1 antibody staining with that of endothelial cell marker vWF and found no significant overlap in staining. This indicated that some PMSCs have a pericyte-like phenotype. We propose that the vascular niche harbours a pool of PMSCs that can give rise to committed progenitors for tissue maintenance and repair, and that PMSCs contribute to vessel maturation and stabilization.

Angiogenesis, a process that newly-formed blood vessels sprout from pre-existing ones, is vital for vertebrate development and adult homeostasis. Previous studies have demonstrated that the neuronal guidance molecule netrin-1 participates in angiogenesis and morphogenesis of the vascular system. Netrin-1 exhibits dual activities in angiogenesis: either promoting or inhibiting angiogenesis. The anti-angiogenic activity of netrin-1 is mediated by UNC5B receptor. However, how netrin-1 promotes angiogenesis remained unclear. Here we report that CD146, an endothelial transmembrane protein of the immunoglobulin superfamily, is a receptor for netrin-1. Netrin-1 binds to CD146 with high affinity, inducing endothelial cell activation and downstream signaling in a CD146-dependent manner. Conditional knockout of the cd146 gene in the murine endothelium or disruption of netrin-CD146 interaction by a specific anti-CD146 antibody blocks or reduces netrin-1-induced angiogenesis. In zebrafish embryos, downregulating either netrin-1a or CD146 results in vascular defects with striking similarity. Moreover, knocking down CD146 blocks ectopic vascular sprouting induced by netrin-1 overexpression. Together, our data uncover CD146 as a previously unknown receptor for netrin-1 and also reveal a functional ligand for CD146 in angiogenesis, demonstrating the involvement of netrin-CD146 signaling in angiogenesis during vertebrate development.

BACKGROUND

The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours.

RESULTS

We isolated an original type of stromal cells, referred to as "Hospicells" from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance.

CONCLUSIONS

This is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient's tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.

BACKGROUND

Adipose stem cells represent a heterogenous population. Understanding the functional characteristics of subpopulations will be useful in developing adipose stem cell-based therapies for regenerative medicine applications. The aim of this study was to define distinct populations within the stromal vascular fraction based on surface marker expression, and to evaluate the ability of each cell type to differentiate to mature adipocytes.

METHODS

Subcutaneous whole adipose tissue was obtained by abdominoplasty from human patients. The stromal vascular fraction was separated and four cell populations were isolated by flow cytometry and studied. Candidate perivascular cells (pericytes) were defined as CD146(+)/CD31(-)/CD34(-). Two CD31(+) endothelial populations were detected and differentiated by CD34 expression. These were tentatively designated as mature endothelial (CD31(+)/CD34(-)), and immature endothelial (CD31(+)/CD34(+)). Both endothelial populations were heterogeneous with respect to CD146. The CD31(-)/CD34(+) fraction (preadipocyte candidate) was also CD90(+) but lacked CD146 expression.

RESULTS

Proliferation was greatest in the CD31(-)/CD34(+) group and slowest in the CD146 group. Expression of adipogenic genes, peroxisome proliferator-activated receptor-γ, and fatty acid binding protein 4, were significantly higher in the CD31(-)/CD34(+) group compared with all other populations after in vitro adipogenic differentiation. This group also demonstrated the highest proportion of AdipoRed lipid staining.

CONCLUSIONS

The authors have isolated four distinct stromal populations from human adult adipose tissue and characterized their adipogenic potential. Of these four populations, the CD31/CD34(+) group is the most prevalent and has the greatest potential for adipogenic differentiation. This cell type appears to hold the most promise for adipose tissue engineering.

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However, no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work, we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF, EGF and PDGF. These cells are SSEA4(+), OCT3/4(+), NANOG(+), SOX2(+), LIN28(+), CD13(+), CD105(+), CD34(-), CD45(-), CD90(+), CD29(+), CD73(+), STRO1(+) and CD146(-), and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly, DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm, endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4, GATA6, MIXL1, NANOG, OCT3/4, SOX1 and SOX2 to determine the degree of similarity between DPPSCs, EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs, hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages, they represent an easily accessible source of stem cells, which opens a range of new possibilities for regenerative medicine.

OBJECTIVE

The main purpose of this study was to isolate and characterize gingival connective tissue-derived mesenchymal stem cells (GMSCs). The secondary purpose was to present a modified isolation method for the GMSCs.

METHODS

Collected healthy gingival tissue samples were de-epithelialized and minced into small fragments. The tissues were digested by dispase and collagenase IV for 30 min. The first digested cell suspension was discarded, and then additional digestion was performed to the remaining cells in the same solution for 90 min. The isolated cells from gingiva was incubated in 37°C humidified condition and observed by inverted microscope. Cytoskeletal morphology was evaluated by phalloidin immunofluorescence. Potency of the cells was tested by colony-forming unit fibroblast assay. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometric, immunofluorescence analysis.

RESULTS

GMSCs showed spindle-shaped, fibroblast-like morphology, colony-forming abilities, adherence to plastic and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency. GMSCs expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 in flow cytometry. Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and a mesenchymal marker (vimentin) were observed by immunofluorescence.

CONCLUSIONS

In conclusion, we isolated and characterized stem cells from human gingival connective tissue with modified protocol. GMSCs showed multipotency with high proliferation and characteristics of mesenchymal stem cells. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs' proliferation and cryopreservation.

Mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of tissues through homing to injured areas, especially to hypoxic, apoptotic, or inflamed areas and releasing factors that hasten endogenous repair. In some cases genetic engineering of the MSC is desired, since they are excellent delivery vehicles. We have derived MSCs from the human embryonic stem cell (hESC) line H9 (H9-MSCs). They expressed CD105, CD90, CD73, and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR, the hESC pluripotency markers SSEA-4 and Tra-1-81, and the hESC early differentiation marker SSEA-1. Marrow-derived MSCs showed a similar phenotype. H9-MSCs did not form teratoma in our initial studies, whereas the parent H9 line did so robustly. H9-MSCs differentiated into bone, cartilage, and adipocytes in vitro, and displayed increased migration under hypoxic conditions. Finally, using a hindlimb ischemia model, H9-MSCs were shown to home to the hypoxic muscle, but not the contralateral limb, by 48 h after IV injection. In summary, we have defined methods for differentiation of hESCs into MSCs and have defined their characteristics and in vivo migratory properties.

When ruptured, the anterior cruciate ligament (ACL) of the human knee has limited regenerative potential. However, the goal of this report was to show that the cells that migrate out of the human ACL constitute a rich population of progenitor cells and we hypothesize that they display mesenchymal stem cell (MSC) characteristics when compared with adherent cells derived from bone marrow or collagenase digests from ACL. We show that ACL outgrowth cells are adherent, fibroblastic cells with a surface immunophenotype strongly positive for cluster of differentiation (CD)29, CD44, CD49c, CD73, CD90, CD97, CD105, CD146, and CD166, weakly positive for CD106 and CD14, but negative for CD11c, CD31, CD34, CD40, CD45, CD53, CD74, CD133, CD144, and CD163. Staining for STRO-1 was seen by immunohistochemistry but not flow cytometry. Under suitable culture conditions, the ACL outgrowth-derived MSCs differentiated into chondrocytes, osteoblasts, and adipocytes and showed capacity to self-renew in an in vitro assay of ligamentogenesis. MSCs derived from collagenase digests of ACL tissue and human bone marrow were analyzed in parallel and displayed similar, but not identical, properties. In situ staining of the ACL suggests that the MSCs reside both aligned with the collagenous matrix of the ligament and adjacent to small blood vessels. We conclude that the cells that emigrate from damaged ACLs are MSCs and that they have the potential to provide the basis for a superior, biological repair of this ligament.

Stem cells are self-renewing multipotent progenitors with the broadest developmental potential in a given tissue at a given time. Normal stem cells in the adult organism are responsible for renewal and repair of aged or damaged tissue. Adult stem cells are present in virtually all tissues and during most stages of development. In this review, we introduce the reader to the basic information about the field. We describe selected stem cell isolation techniques and stem cell markers for various stem cell populations. These include makers for endothelial progenitor cells (CD146/MCAM/MUC18/S-endo-1, CD34, CD133/prominin, Tie-2, Flk1/KD/VEGFR2), hematopoietic stem cells (CD34, CD117/c-Kit, Sca1), mesenchymal stem cells (CD146/MCAM/MUC18/S-endo-1, STRO-1, Thy-1), neural stem cells (CD133/prominin, nestin, NCAM), mammary stem cells (CD24, CD29, Sca1), and intestinal stem cells (NCAM, CD34, Thy-1, CD117/c-Kit, Flt-3). Separate section provides a concise summary of recent clinical trials involving stem cells directed towards improvement of a damaged myocardium. In the last part of the review, we reflect on the field and on future developments.

OBJECTIVE

Pericytes are critical cellular components of the microvasculature that play a major role in vascular development and pathologies, yet their study has been hindered by lack of a standardized method for their isolation and growth. Here we report a method for culturing human pericytes from a readily available tissue source, placenta, and provide a thorough characterization of resultant cell populations.

METHODS

We developed an optimized protocol for obtaining pericytes by outgrowth from microvessel fragments recovered after enzymatic digestion of human placental tissue. We characterized outgrowth populations by immunostaining, by gene expression analysis, and by functional evaluation of cells implanted in vivo.

RESULTS

Our approach yields human pericytes that may be serially expanded in culture and that uniformly express the cellular markers NG2, CD90, CD146, alpha-SMA, and PDGFR-beta, but lack markers of smooth muscle cells, endothelial cells, and leukocytes. When co-implanted with human endothelial cells into C.B-17 SCID/bg mice, human pericytes invest and stabilize developing human endothelial cell-lined microvessels.

CONCLUSIONS

We conclude that our method for culturing pericytes from human placenta results in the expansion of functional pericytes that may be used to study a variety of questions related to vascular biology.

OBJECTIVE

We examined whether RNA expression of CD133, a surface molecule expressed on progenitors from hematopoietic and endothelial lineages, and CD146, a pan-endothelial marker, are increased in the blood of cancer patients and whether these factors correlate with patient characteristics and are predictive factors of survival.

METHODS

We developed a real-time quantification method (nuclear acid sequence-based amplification) to determine expression of CD146 and CD133 mRNA in the peripheral blood mononuclear cells of 131 progressive cancer patients, 37 healthy volunteers, and 5 patients who received granulocyte colony-stimulating factor. Overall survival and other clinicopathologic variables were obtained. Cox proportional hazards studies were done.

RESULTS

We show that patients with metastatic disease have a significant increase in CD133 mRNA (P = 0.03), specifically patients with bone metastasis (P < 0.001). Cancer patients with high CD133 mRNA expression, using a defined cutoff value, show a decreased survival compared with patients with low or undetectable CD133 expression (21% versus 45% cumulative survival, respectively, after 20 months; P = 0.01). Among patients with metastasis to the bone, cumulative survival was 22%, compared with 61% for patients with high or low CD133 levels (P = 0.004). Multivariate analysis showed that CD133 expression is an independent predictor for overall survival in patients with bone metastases. CD146 mRNA was not increased in patients with cancer, nor did it correlate with clinical variables or survival.

CONCLUSIONS

CD133, but not CD146, mRNA expression is increased in cancer patients with metastatic disease, specifically with bone metastasis. In addition, CD133 mRNA expression seems to be an independent prognostic factor for overall survival.