OBJECTIVETo investigate the expression of chemokine stromal cell-derived factor-1 (SDF-1) receptor CXCR4 in human gingival mesenchymal stem cells (GMSCs) and the migration potential of GMSCs stimulated with SDF-1.

METHODSHuman GMSCs were isolated by single-cell cloning method. Their cell surface markers were characterized by flow cytometry, and the rate of colony formation was evaluated. Differentiation assay was used to detect the differentiation potential of GMSCs. The expression of chemokine SDF-l receptor CXCR4 in GMSCs was detected by immunocytochemical staining. The chemotactic effect of SDF-1 on GMSCs was detected using a 24-multiwell Transwell cell culture chamber. The number of net migrated cells was counted in different microscope fields.

RESULTSHuman GMSCs possessed high self-renewal potential and formed single-cell colonies cultured in vitro. GMSCs expressed mesenchymal stem cells-associated markers CD44, CD73, CD90, CD105, and CD166, and the expression of hemopoietic stem cell surface markers CD14, CD34, and CD45 was negative. GMSCs differentiated into osteoblasts and adipocytes under defined culture conditions. The colony forming unit-fibroblastic for GMSCs was 21.4%/±2.8%. Immunocytochemical staining demonstrated that GMSCs expressed chemokine SDF-1 receptor CXCR4. The number of GMSCs migrating at concentrations of 100 ng.mL-1 and 200 ng.mL-1 of SDF-l in the Transwell cell culture chamber was significantly higher than that of the negative control (189.3±4.4, 164.6±4.9 cells/field vs. 47.8±2.5 cells/field, P<0.01). Treatment with the CXCR4 neutralizing antibody, an antagonist for CXCR4, significantly reduced the migratory effect compared with the negative controls (29.0±2.4 cells/field vs. 47.8±2.5 cells/field, P<0.01).

CONCLUSIONHuman GMSCs express chemokine SDF-l receptor CXCR4. SDF-1 may participate in regulating chemotaxis of human GMSCs. Results suggest that the migration induced by SDF-1 is mediated by CXCR4.

Adipocytes ; Cell Culture Techniques ; Cell Differentiation ; Chemokine CXCL12 ; metabolism ; Chemotaxis ; Flow Cytometry ; Gingiva ; physiology ; Humans ; Mesenchymal Stromal Cells ; physiology ; Osteoblasts ; Receptors, CXCR4 ; Signal Transduction

OBJECTIVETo investigate the chemotactic response of human periodontal ligament stem cells (PDLSCs) to bone morphogenetic protein-2 (BMP-2).

METHODSHuman PDLSCs were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate PDLSCs by limited dilution method. The expression of Vimentin and stem cell marker STRO-1 on PDLSCs were demonstrated with immunocytochemical staining. Differentiation assay was used to detect the differentiation potential of PDLSCs. Cloning formation experiment and 5-bromo-2-deoxyuridine (BrdU) incorporation assay were used to determine the stem cell characteristics of PDLSCs. The chemotactic effect of BMP-2 on PDLSCs was detected by using a 24-multiwell Transwell cell culture chamber. The number of net migrated cells was counted in different microscope fields.

RESULTSHuman PDLSCs displayed positive staining for Vimentin and expressed the stem cell marker STRO-1. These cells differentiated into osteoblasts and adipocytes under defined culture conditions, possessed high self-renewal potential and formed single-cell colonies in vitro. The number of cells migrating at concentrations of 100, 200 ng mL(-1) of BMP-2 in Transwell cell culture chamber was significantly higher than that of negative control (P<0.01).

CONCLUSIONBMP-2 may participate in regulating chemotaxis of human PDLSCs.

Adipocytes ; Bone Morphogenetic Protein 2 ; Cell Culture Techniques ; Cell Differentiation ; Humans ; Osteoblasts ; Periodontal Ligament ; Stem Cells

ObjectiveTo?investigate?the?expression?of?chemokine?stromal?cell-derived?factor-1?(SDF-1)?receptor?CXCR4?in?human?gingival?mesenchymal?stem?cells?(GMSCs)?and?the?migration?potential?of?GMSCs?stimulated?with?SDF-1.?Methods?Human?GMSCs?were?isolated?by?single-cell?cloning?method.?Their?cell?surface?markers?were?characterized?by?flow?cytometry,?and?the?rate?of?colony?formation?was?evaluated.?Differentiation?assay?was?used?to?detect?the?differentiation?potential?of?GMSCs.?The?expression?of?chemokine?SDF-1?receptor?CXCR4?in?GMSCs?was?detected?by?immunocytochemical?staining.?The?che-motactic?effect?of?SDF-1?on?GMSCs?was?detected?using?a?24-multiwell?Transwell?cell?culture?chamber.?The?number?of?net?migrated?cells?was?counted?in?different?microscope?fields.?Results???Human?GMSCs?possessed?high?self-renewal?potential?and?formed?single-cell?colonies?cultured?in vitro.?GMSCs?expressed?mesenchymal?stem?cells-associated?markers?CD44,?CD73,?CD90,?CD105,?and?CD166,?and?the?expression?of?hemopoietic?stem?cell?surface?markers?CD14,?CD34,?and?CD45?was?negative.?GMSCs?differentiated?into?osteoblasts?and?adipocytes?under?defined?culture?conditions.?The?colony?forming?unit-fibroblastic?for?GMSCs?was?21.4%±2.8%.?Immunocytochemical?staining?demonstrated?that?GMSCs?expressed?chemokine?SDF-1?receptor?CXCR4.?The?number?of?GMSCs?migrating?at?concentrations?of?100?ng·mL-1?and?200?ng·mL-1?of?SDF-1?in?the?Transwell?cell?culture?chamber?was?significantly?higher?than?that?of?the?negative?control?(189.3±4.4,?164.6±4.9?cells/field?vs.?47.8±2.5?cells/field,?P<0.01).?Treatment?with?the?CXCR4?neutralizing?antibody,?an?antagonist?for?CXCR4,?significantly?reduced?the?migratory?effect?compared?with?the?negative?con-trols?(29.0±2.4?cells/field?vs.?47.8±2.5?cells/field,?P<0.01).?Conclusion???Human?GMSCs?express?chemokine?SDF-1?receptor?CXCR4.?SDF-1?may?participate?in?regulating?chemotaxis?of?human?GMSCs.?Results?suggest?that?the?migration?induced?by?SDF-1?is?mediated?by?CXCR4.

OBJECTIVETo study the pattern of CGG repeat instability within germline cells derived from two male fetuses affected with Fragile X syndrome (FXS).

METHODSThe length and methylation status of CGG repeats within the testes of a fetus carrying a full FXS mutation and another fetus carrying mosaicism FXS mutation were analyzed with Southern blotting and AmplideX FMR1 PCR. Immunohistochemistry was also applied for the measurement of FMR1 protein (FMRP) expression within the testes.

RESULTSFor the fetus carrying the full mutation, Southern blotting analysis of the PCR product has detected an expected band representing the full mutation in its brain and a premutation band of > 160 CGG repeats in its testis. Whereas the pattern of premutation/full mutation in mosaic testis was similar to that in peripheral blood and no sign of contracted fragment was found other than a band of about 160 CGG repeats. Immunohistochemistry assay with a FMRP-specific antibody demonstrated a number of FMRP-positive germ cells, which suggested a contraction from full mutation to premutation alleles.

CONCLUSIONThis study has clarified the instability pattern of CGG repeat and expression of FMRP protein within the testes of fetuses affected with FXS, confirming that the CGG repeat can contract progressively within the germline. The FMRP expression in the testis is consistent with spermatogonium proliferation, and thus the contraction from full mutation to unmethylated premutations may occur for the requirement of FMRP expression during spermatogenesis. The better understanding of FMRP function during germ cell proliferation may elucidate the mechanism underlying the contraction of full FXS mutation in male germline.

Abortion, Eugenic ; Blotting, Southern ; Brain ; embryology ; metabolism ; DNA Methylation ; Fatal Outcome ; Fetus ; cytology ; metabolism ; Fragile X Mental Retardation Protein ; genetics ; metabolism ; Fragile X Syndrome ; diagnosis ; genetics ; Humans ; Immunohistochemistry ; Male ; Mosaicism ; Mutation ; Polymerase Chain Reaction ; Spermatozoa ; metabolism ; Testis ; cytology ; embryology ; metabolism ; Trinucleotide Repeat Expansion ; genetics

Objective To compare the CGG-repeat-length and its methylation status in fetal tissues and to explicate the heterogeneity of CGG repeats.Method Multiple tissues from a full mutation (August 2013) and a mosaic aborted fetus of 23-week gestation(May 2012) were collected and genomic DNA from these tissues was extracted.The CGG-repeat-length and methylation status in fetal tissues were determined by a combined strategy of Southern blotting and GC-Rich PCR.FMR1 expression was measured by real time PCR and Western blotting.Result CGG-repeat-length in different tissues of each fetus was similar.A major methylated band in the full mutation range (540 CGG repeats) was detected in the brain,skin,testis and kidney tissues of Case 1.An unmethylated premutation band with 160 CGG repeats,and another two bands with 470 and 1 100 CGG repeats in the full mutation range were shown in the brain,skin,testis,lung,stomach,gut,liver,kidney,heart and blood of Case 2.However,the methylation status of CGG repeats in the mosaic fetus was heterogeneous among different tissues.The lowest premutation ratio was in the brain of the mosaic fetus compared with other tissues,and correspondingly FMR1 expression in its brain was minimum.Conclusion This study clarify the tissue heterogeneity of CGG repeats and provides information for the genetic counseling and clinical diagnosis in fragile X syndrome.Based on the fact that the mosaic fetus' mother is a carrier of full mutation,it is speculated that the maternal CGG repeat has contracted before the differentiation of trilaminar germ disc.