OBJECTIVETo identify the existence of the dental pulp stem cells in Beagle's pulp tissue by using the same methods of isolating and culturing the human dental pulp stem cells.

METHODSPulp tissue was extirpated from the crown and root of the Beagle's healthy permanent tooth, and digested by dispase for cell culture. Classical identification methods of mesenchymal stem cells including observation of biological characteristics, capacity of multilineage differentiation, and expression of specific markers associated with mesenchymal stem cells were applied to verify the existence of Beagle's dental pulp stem cells.

RESULTSA clonogenic, rapidly proliferative population of cells were isolated from Beagle' pulp tissue. Under the same culture condition, the Beagle's dental pulp stem cells had a significant higher colony-forming unit-fibroblast (CFU-F) formation rate (150 colony/10(4) cells) than the dental pulp cells derived from the human pulp tissue (60 colony/10(4) cells). These cells also had the multilineage differentiation ability. They could be induced to form mineralized nodules, lipid droplets and chondrocytes. Furthermore these cells expressed the mesenchymal stem cell markers including STRO-1, CD146, alkaline phosphatase, nestin, vimentin and cytokeratin-18.

CONCLUSIONSThere are dental pulp stem cells in the Beagle's pulp tissue.

Alkaline Phosphatase ; metabolism ; Animals ; Antigens, Surface ; metabolism ; CD146 Antigen ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Colony-Forming Units Assay ; Dental Pulp ; cytology ; Dogs ; Humans ; Keratin-18 ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Nestin ; metabolism ; Osteogenesis ; Vimentin ; metabolism

BACKGROUNDRecombinant human endostatin (rh-endostatin, Endostar) has been proved to be an inhibitor of angiogenesis. Docetaxel has been also considered as a common chemotherapeutic agent with inhibition of angiogenesis of malignancies. However, their function has been seldom compared and a best synergism protocol is not determined. This study aimed to compare the effects of two drugs, investigate their combined impact on human umbilical vein endothelial cells (HUVECs), a molecular basis and find ideal protocols to inhibit endothelial cell proliferation.

METHODSHUVECs on confluent growth or activated by vascular endothelial growth factor (VEGF) were treated by rh-endostatin or/and docetaxel at respective gradient concentration in following operations as cell proliferation determined by MTT assay, cell cycle distribution, apoptosis and markers of CD146, CD62E and CD105 detected by flow cytometery, the structure of the channel formed by HUVECs measured by tube formation count.

RESULTSRh-endostatin exhibited time dependent inhibition of proliferation while docetaxel showed both time and dose dependent inhibition. HUVECs accumulated in G(0)-G(1) with decreased numbers of cells in G(2) after a single treatment of rh-endostatin or that followed by docetaxel treatment. Cells accumulated in G(2) after both a single docetaxel and simultaneous administration. Both the number of cells in G(0)-G(1) and apoptotic cells were increased by docetaxel followed by rh-endostatin treatment. The number of non-apoptotic cells at G(0)-G(1) was increased by first administering rh-endostatin then docetaxel. Sequential treatment of docetaxel followed by rh-endostatin resulted in the greatest increase in apoptosis (34.7%) and the second highest apoptosis was seen with simultaneous administration (18.2%). Expression of CD146 and CD105 on confluent HUVECs was reduced at certain doses of rh-endostatin and/or docetaxel. However, rh-endostatin reduced CD105 without any apparent impact on either CD146 or CD62E expression, whereas these markers were down-regulated by docetaxel after pre-activation by VEGF. Rh-endostatin treatment maintained tube-like structures for a limited time. In contrast, docetaxel swiftly reduced tube formation. Simultaneous treatment, or docetaxel followed by rh-endostatin, exhibited a stronger inhibition on tube formation than either agent alone.

CONCLUSIONSBoth rh-endostatin and docetaxel can inhibit HUVEC proliferation while the high apoptotic rate after combined administration was probably owing to different sequent administration by docetaxel followed by rh-endostatin or simultaneous treatment. Both proliferation and adhesion molecules on HUVECs of confluent growth are down-regulated by the two drugs. The rh-endostatin decreased proliferation markers, but only slightly modified adhesion molecules, while both markers were down-regulated by docetaxel on HUVECs activated by VEGF. Rh-endostatin could maintain adhesion of HUVECs at first then induce cells apoptosis to damage tube formation. We hypothesize that it could lead to vascular normalization in short time. In contrast, docetaxel can suppress HUVEC proliferation, adhesion, and reduced tube formation swiftly due to its cytotoxicity. Combined treatments can induce a synergistic inhibition of tube formation.

Antigens, CD ; metabolism ; Apoptosis ; drug effects ; CD146 Antigen ; metabolism ; Cell Proliferation ; drug effects ; E-Selectin ; metabolism ; Endoglin ; Endostatins ; pharmacology ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Receptors, Cell Surface ; metabolism ; Recombinant Proteins ; pharmacology ; Taxoids ; pharmacology

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.
Adult ; Adult Stem Cells ; cytology ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; Biomarkers ; analysis ; CD146 Antigen ; analysis ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Dental Pulp ; cytology ; Flow Cytometry ; methods ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Nerve Tissue Proteins ; analysis ; Odontogenesis ; physiology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis

OBJECTIVETo assess the effect of endostatin on growth and neoplastic angiogenesis in transplanted human lung adenocarcinoma Calu-6 tumor in nude mice.

METHODSTo treat Calu-6 tumor-bearing mice with endostatin at different doses, and to record the changes of the tumor size. The expressions of survivin, VEGF, COX-2 and MVD in tumor tissue were examined by immunohistochemistry staining, circulating endothelial cells (CECs) by flow cytometry and mRNA of CD146 and CD105 by RT-PCR and real-time PCR.

RESULTSAfter endostatin treatment, the tumor size was conspicuously shrunk, and the expressions of survivin, COX-2 and VEGF protein and MVD in tumor tissue decreased concomitantly with the significant difference between each of trial groups and control group (all P < 0.05). Both CECs and mRNA of CD146 and CD105 diminished remarkably. A positive correlation between both exhibition and change of amount of activated CECs and survivin, VEGF expression and MVD count in tumor tissue was found.

CONCLUSIONEndostatin can decrease the expression of survivin, COX-2, VEGF and MVD, and to inhibit the growth of transplanted tumor. Activated CECs may probably serve as an ideal marker to predict the efficacy and prognosis of anti-angiogenesis therapy.

Adenocarcinoma ; pathology ; Angiogenesis Inhibitors ; administration & dosage ; pharmacology ; Animals ; Antigens, CD ; metabolism ; Antineoplastic Agents ; administration & dosage ; pharmacology ; CD146 Antigen ; metabolism ; Cell Line, Tumor ; Cyclooxygenase 2 ; metabolism ; Dose-Response Relationship, Drug ; Endoglin ; Endostatins ; administration & dosage ; pharmacology ; Endothelial Cells ; pathology ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; Lung Neoplasms ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microtubule-Associated Proteins ; metabolism ; Microvessels ; pathology ; Neoplasm Transplantation ; RNA, Messenger ; metabolism ; Random Allocation ; Receptors, Cell Surface ; metabolism ; Tumor Burden ; drug effects ; Vascular Endothelial Growth Factor A ; metabolism

Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51(+)/CD140α(+), 0.8% were CD271(+), and 2.4% were STRO-1(+)/CD146(+). Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271(+) DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.
Adaptor Proteins, Signal Transducing ; analysis ; Adult ; Aggrecans ; analysis ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; CD146 Antigen ; analysis ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Collagen Type II ; analysis ; Core Binding Factor Alpha 1 Subunit ; analysis ; Flow Cytometry ; methods ; Homeodomain Proteins ; analysis ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; physiology ; Multipotent Stem Cells ; cytology ; physiology ; Nerve Tissue Proteins ; analysis ; Osteogenesis ; physiology ; Periodontal Ligament ; cytology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis ; SOX9 Transcription Factor ; analysis ; Time Factors ; Transcription Factors ; analysis

BACKGROUND AND OBJECTIVELeukemic microenvironment has a major role in the progression of leukemia. Leukemic cells can induce reversible changes in microenvironmental components, especially the stromal function which results in improved growth conditions for maintaining the malignant leukemic cells. This study aimed to investigate the survival advantage of leukemic cells over normal hematopoietic cells in stromal microenvironment in long term.

METHODSThe mice were injected intraperitoneally with N-N' ethylnitrosourea (ENU) to induce leukemia; the mice received injection of normal saline were used as control. At 180 days after ENU induction, the mice were killed and the bone marrows were cultured for 19 days. Colony-forming assays were used to analyze the formation of various cell colonies. The expression of Sca-1, CD146, VEGFR2, CD95, pStat3, pStat5, and Bcl-xL in marrow cells were detected by flow cytometry.

RESULTSLong-term leukemic bone marrow culture showed abnormal elongated stromal fibroblasts with almost absence of normal hematopoietic cells. Adherent cell colonies were increased, but CFU-F and other hematopoietic cell colonies were significantly decreased in leukemia group (P<0.001). Primitive progenitor-specific Sca-1 receptor expression was decreased with subsequent increased expression of CD146 and VEGFR-2 in leukemic bone marrow cells. Decreased Fas antigen expression with increased intracellular pStat3, pStat5 and Bcl-xL proteins were observed in leukemic bone marrow cells.

CONCLUSIONSStromal microenvironment shows altered morphology and decreased maturation in leukemia. Effective progenitor cells are decreased in leukemia with increased leukemia-specific cell population. Leukemic microenvironment plays a role in promoting and maintaining the leukemic cell proliferation and survivability in long term.

Animals ; Antigens, Ly ; metabolism ; Bone Marrow Cells ; metabolism ; pathology ; CD146 Antigen ; metabolism ; Cell Count ; Cells, Cultured ; Colony-Forming Units Assay ; Erythroid Precursor Cells ; metabolism ; pathology ; Ethylnitrosourea ; Female ; Fibroblasts ; metabolism ; pathology ; Granulocyte-Macrophage Progenitor Cells ; metabolism ; pathology ; Granulocytes ; metabolism ; pathology ; Hematopoiesis ; Hematopoietic Stem Cells ; metabolism ; pathology ; Leukemia ; chemically induced ; metabolism ; pathology ; Male ; Membrane Proteins ; metabolism ; Mice ; Myeloid Progenitor Cells ; metabolism ; pathology ; Phenotype ; STAT3 Transcription Factor ; metabolism ; STAT5 Transcription Factor ; metabolism ; Tumor Microenvironment ; physiology ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; bcl-X Protein ; metabolism ; fas Receptor ; metabolism

CD146 Antigen ; immunology ; Fibroblasts ; immunology ; pathology ; Humans ; Scleroderma, Systemic ; drug therapy ; immunology ; pathology

CD146 Antigen ; metabolism ; Humans ; Pericytes ; cytology ; metabolism ; Receptor, Platelet-Derived Growth Factor beta ; metabolism

OBJECTIVE: Stem cells from human exfoliated teeth (SHED) were sorted by magnetically activated cell sorting (MACS) technique to obtain the CD146 positive and negative cell subpopulation. Then the biological characteristics of these subpopulations were compared to explore their specific application potential in tissue engineering. METHODS: In this study, freshly extracted deciduous teeth without any caries or dental pulp disease were obtained. SHED was isolated using enzyme digestion method and then sorted by MACS, CD146 positive cells and CD146 negative cells were obtained after cell sorting. The biological characteristics of the unsorted mixed cells, CD146 positive subpopulation and CD146 negative subpopulation were compared. The proliferation ability was detected through cell counting kit-8 (CCK-8) and colony-forming unit (CFU). After osteogenic induction, alizarin red staining was performed and the gene expression of osteogenic related markers was detected by quantitative real-time polymerase chain reaction(qPCR). After adipogenic induction, oil-red O staining was performed and the gene expression of adipogenic related markers was detected. After neurogenic differentiation induction, the expression of neural markers was detected by immunofluorescence and the gene expression of neural markers was detected by qPCR. RESULTS: SHED of the fifth passage was sorted by MACS. And the CD146 positive cell subpopulation and CD146 negative cell subpopulation were obtained. CCK8 assay showed that the proliferative tendency of the three cell groups was consistent, but the proliferation potential of CD146 positive and negative cell subpopulations was significantly lower than that of the unsorted cells. The colony forming rates of the unsorted mixed cell group, CD146 positive and negative populations were 28.6%±3%,17.1%±2.3% and 27.5%±2.5%, respectively. After 21 days of osteogenic induction, alizarin red staining and qPCR showed that the CD146 positive cell population had more mineralized nodule formation and expressed higher level of osteogenic related genes compared with the other two groups. After 21 days of adipogenic induction, oil red O staining and qPCR results showed that the CD146 negative subpopulation produced more lipid droplets and the expression of lipid related genes increased more significantly. After 14 days of neural induction, cell immunofluorescence and qPCR results showed that the unsorted mixed cell group and CD146 positive subpopulation expressed glial cell marker, and the expressions of neural precursor cells and neuronal marker increased significantly in negative subpopulation. CONCLUSION The unsorted mixed cells showed better proliferative potential than CD146 positive and negative subpopulations. The CD146 positive subpopulation was most potent in osteogenic differentiation; it was more suitable for bone tissue engineering. The CD146 negative cells had stronger adipogenic differentiation potential than the other two cell groups; different subpopulations differed in neural differentiation.
Bone and Bones ; CD146 Antigen/analysis* ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stem Cells ; Neural Stem Cells ; Neurons ; Osteogenesis ; Staining and Labeling ; Tissue Engineering ; Tooth, Deciduous/cytology*

OBJECTIVETo study the clinicopathologic features and immunophenotype of placental site trophoblastic tumor (PSTT) and epithelioid trophoblastic tumor (ETT).

METHODSDuring the period from 1959 to 2005, a total of 1012 cases of gestational trophoblastic disease were diagnosed in Beijing Obstetrics and Gynecology Hospital. Six cases of PSTT and a case of ETT were retrieved from the archives of Beijing Obstetrics and Gynecology Hospital. Immunohistochemical study for cytokeratin 18, human chorionic gonadotropin (hCG), human placental lactogen (hPL), Mel-CAM (CD146), placental-like alkaline phosphatase (PLAP), epithelial membrane antigen (EMA), inhibin-alpha and proliferative cell nuclear antigen (PCNA) were performed. The morphologic features and immunohistochemical findings were compared with those of the controlled group which consisted of 20 cases of early gestational villi with decidua basalis and 20 cases of hydatidiform moles with implantation site.

RESULTSThe mean age of patients with PSTT was 32.4, while the age of patients with ETT was 36. Major clinical findings included irregular vaginal bleeding and amenorrhea. Preoperative serum hCG level varied from normal to moderately elevated. Serum testosterone level was raised in 1 case. Uterine curettage could achieve an accurate pathologic diagnosis in 60% of cases. ETT involved mainly the lower uterine segment and endocervix. Histologically, PSTT cells permeated between the myometrial fibers and vessels either individually or connecting in cords or sheets in a manner reminiscent of the implantation site reaction. ETT composed of a relatively uniform population of mononuclear trophoblastic cells, clumping together in nests as the cell islets associating with eosinophilic, fibrillary and hyaline material and necrotic debris, forming a "geographic map" like pattern. Immunohistochemical study for hPL, hCG, Mel-CAM (CD146) and PLAP was most helpful for the differential diagnosis. The duration of follow-up varied from 14 months to 19 years. One case of PSTT developed metastasis in pancreas, 5 months after the operation. The remaining patients survived without tumor recurrence.

CONCLUSIONSPSTT is a tumor of implantation site intermediate trophoblasts while ETT differentiates towards chorionic-type intermediate trophoblasts. The different pathologic features and immunophenotype observed were closely related with the difference in tumor cell differentiation. An accurate pathologic diagnosis of the uterine curettage material is important for the clinical management. According to the limited follow-up data available, the clinical behavior of ETT is seemed similar to that of PSTT.

Adult ; Alkaline Phosphatase ; metabolism ; CD146 Antigen ; metabolism ; Chorionic Gonadotropin ; metabolism ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Humans ; Hysterectomy ; methods ; Immunohistochemistry ; Isoenzymes ; metabolism ; Middle Aged ; Placental Lactogen ; metabolism ; Pregnancy ; Prognosis ; Trophoblastic Neoplasms ; metabolism ; pathology ; surgery ; Trophoblastic Tumor, Placental Site ; metabolism ; pathology ; surgery ; Uterine Neoplasms ; metabolism ; pathology ; surgery