OBJECTIVETo isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.

METHODSWe detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.

RESULTSThe isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.

CONCLUSIONCD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

Adult Stem Cells ; cytology ; Biomarkers ; metabolism ; Cell Separation ; methods ; Cell Shape ; Cell Size ; Coculture Techniques ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; metabolism ; Humans ; Immunohistochemistry ; Male ; Receptors, G-Protein-Coupled ; metabolism ; Sertoli Cells ; Spermatogonia ; cytology ; Testis ; metabolism ; Thy-1 Antigens ; isolation & purification ; metabolism ; Ubiquitin Thiolesterase ; metabolism

OBJECTIVETo confirm the malignant phenotype of hepatocarcinoma cell (HCC) lines at various stages of differentiation (MHCC97L, MHCC97H and HCCLM3) and to explore their expression levels of cancer stem cell (CSC) markers.

METHODSThe invasive and proliferative properties of each HCC line were assessed by transwell assay and the Cell Counting Kit-8 (CCK-8) colorimetric assay. Sensitivity to chemotherapy was assessed by treatment with oxaliplatin and determination of the half inhibitory concentration (IC50). The expression of CD90, EpCAM and CD24 was measured by flow cytometry.

RESULTSThe number of cells that migrated through the invasion assay membrane were significantly different between the three HCC lines: HCCLM3 (30.57 +/- 8.95) more than MHCC97H (21.33 +/- 4.17) more than HCC97L (9.33 +/- 3.85), P less than 0.01. The IC50 was significantly different between the three HCC lines: HCCLM3 (36.57 +/- 6.95) mumol/L more than MHCC97H (26.35+/-3.88) mumol/L more than MHCC97L (17.68 +/- 3.25) mumol/L. The CSC marker with the highest expression on all three HCC lines was CD90 (HCCLM3: 0.92% +/- 0.21%, MHCC97H: 1.98% +/- 0.23%, and MHCC97L: 2.55% +/- 0.34%), followed by EpCAM (2.11% +/- 0.32%, 3.23% +/- 0.18%, and 4.38% +/-0.49%, respectively), and CD24 as the lowest (0.68% +/- 0.37%, 1.22% +/- 0.26%, and 1.36% +/- 0.24%, respectively).

CONCLUSIONHigher expression of CSC markers on HCC lines is associated with a stronger invasive ability and higher sensitivity to chemotherapy.

Antigens, Neoplasm ; metabolism ; CD24 Antigen ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Adhesion Molecules ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Epithelial Cell Adhesion Molecule ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Neoplastic Stem Cells ; cytology ; metabolism ; Signal Transduction ; Thy-1 Antigens ; metabolism

OBJECTIVETo study the immunophenotype and chromosome karyotype characteristics of leukemic stem cells (LSC) in Uyghur leukemia pediatric patients.

METHODSThe morphological features of LSC in culture in vitro was observed by flow cytometry. The immunophenotype was assessed by detective flow cytometry. The chromosome karyotype was analyzed by R-banding technique.

RESULTSThe LSC showed suspended floating colonies growing in the culture medium, and grew well and proliferated constantly in culture over 8 months. Among the 13 children with AML, there were 10 CD34(+)CD38(-)CD123(+) and CD33(+) cases, 10 CD44(+) cases, 10 CD96(+) cases, and 5 CD90(+) cases. Among the 13 children with B-ALL, there were 6 CD34(+)CD20(-)CD19(+) cases, 7 CD9(+) cases, and 5 CD123(+) cases. Among the 9 children with acute T lymphoblastic leukemia (T-ALL), there were 5 CD34(+)CD7(-) and CD90(+) cases, and 4 CD123(+) cases. Among the 13 cases of AML, 5 cases showed chromosome translocation t(15;17), one case chromosome translocation t(8;21), and 7 cases showed no chromosome karyotype abnormality. Among the 22 ALL cases, there were chromosome translocation t(12;21) in 1 case, t(9;22) in 3 case, hyperdiploid in 2 cases, and 16 cases without karyotype abnormalities. Twenty-nine children received induction remission therapy. Among them, 12 died, including 9 CD96(-)positive cases and 3 CD96(-)negative cases, with a statistically significant difference (P < 0.05).

CONCLUSIONSThe LSC of Uyghur leukemia pediatric patients in Xinjiang express CD9 and CD19 in ALL, and express CD123 and CD90 simultaneously in ALL and AML. The expression of CD96 is one of factors of poor prognosis.

Adolescent ; Antigens, CD ; metabolism ; Antigens, CD19 ; metabolism ; Child ; China ; ethnology ; Diploidy ; Humans ; Immunophenotyping ; Interleukin-3 Receptor alpha Subunit ; metabolism ; Karyotyping ; Leukemia, Myeloid, Acute ; drug therapy ; genetics ; immunology ; pathology ; Neoplastic Stem Cells ; immunology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; immunology ; pathology ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; immunology ; pathology ; Remission Induction ; Tetraspanin-29 ; metabolism ; Thy-1 Antigens ; metabolism ; Translocation, Genetic

OBJECTIVETo investigate whether FTY720 inhibits rat mesangial proliferation and extracellular matrix expansion through suppression of transforming growth factor β1-connective tissue growth factor (TGFβ1-CTGF) pathway, and to explore experimental evidence for its effect on mesangial proliferative glomerulonephritis.

METHODSA rat model of anti-Thy-1 mesangial proliferative glomerulonephritis was established and FTY720 intervention was performed. Periphery blood lymphocyte count, urine protein excretion, glomerular mesangial proliferation, protein and gene expression of TGFβ1 and CTGF and extracellular matrix protein including fibronectin, laminin and collagen IV in isolated glomeruli were documented at 1, 3 and 7 days after injection of anti-Thy-1 antibody.

RESULTSThe model group developed proteinuria at 1, 3 and 7 days after injection of anti-Thy-1 antibody, which were significantly higher [(27.9 ± 7.3), (63.5 ± 18.8) and (52.4 ± 15.4)mg/d, respectively] than those in the control group [(8.4 ± 2.4), (8.4 ± 2.1) and (10.4 ± 3.2) mg/d; respectively, P < 0.01]. FTY720 intervention group showed significantly decreased proteinuria at 3 and 7 days after injection [(31.4 ± 7.0), (25.5 ± 7.7) mg/d, respectively] than model group (P < 0.01), although higher than the control group (P < 0.01). After intervention for 3 and 7 days, FTY720 significantly down-regulated both TGFβ1 and CTGF gene and protein expression in cultured glomeruli, and suppressed the production of glomerular extracellular matrix protein secretion, leading to attenuated mesangial cell proliferation and extracellular matrix expansion in rat anti-Thy-1 mesangial proliferative glomerulonephritis.

CONCLUSIONFTY720 significantly attenuates mesangial proliferation and extracellular matrix expansion through inhibition of TGFβ1-CTGF pathway in rat, and thus ameliorates the development of anti-Thy-1 mesangial proliferative glomerulonephritis.

Animals ; Cell Proliferation ; Connective Tissue Growth Factor ; genetics ; metabolism ; Down-Regulation ; Extracellular Matrix Proteins ; metabolism ; Fingolimod Hydrochloride ; Gene Expression ; Glomerular Mesangium ; metabolism ; pathology ; Glomerulonephritis, Membranoproliferative ; immunology ; metabolism ; pathology ; Immunosuppressive Agents ; pharmacology ; Isoantibodies ; immunology ; Kidney Glomerulus ; metabolism ; pathology ; Male ; Propylene Glycols ; pharmacology ; Proteinuria ; urine ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Sphingosine ; analogs & derivatives ; pharmacology ; Thy-1 Antigens ; immunology ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo study the expression of neonatal Fc receptor in podocytes in human nephritis and immune-induced rat nephritis models: anti-Thy1.1 nephritis and Heymann nephritis.

METHODSThirty-nine cases of renal biopsies were enrolled from September 2009 to February 2010, including 8 cases of minimal change disease, 4 cases of focal segmental glomerulosclerosis, 9 cases of membranous nephropathy, 12 cases of IgA nephropathy and 6 cases of lupus nephritis. Five normal kidney tissue samples adjacent to renal clear-cell carcinoma were served as normal controls. Laser capture microdissection and real-time RT-PCR were used to assess the expression level of FcRn mRNA in glomeruli of various glomerulonephritides, and immunohistochemistry (IHC) of FcRn by SuperVision method was performed. In addition, rat models of mesangial proliferative nephritis (anti-Thy1.1 nephritis) and passive membranous nephropathy (Heymann nephritis) were established and FcRn was examined in renal tissues by IHC.

RESULTSThe FcRn mRNA level in lupus nephritis was statistically higher than that of normal controls (P < 0.05). FcRn protein expression by IHC was seen in lupus nephritis (6/6), membranous nephropathy (6/9) and IgA nephropathy (7/12), significantly higher than that of normal controls (0/5), P < 0.05. Minimal change disease and focal segmental glomerular sclerosis showed minimal or none expression of FcRn (1/8, 0/4 respectively) and not statistically difference from that of normal controls. Furthermore, FcRn expression in podocytes was detected in rat anti-Thy1.1 (3/5) and Heymann nephritis models (2/7) but was not detected in normal controls.

CONCLUSIONSExpression of FcRn in podocytes was up-regulated in immune-induced human nephritis and rat nephritis models of anti-Thy1.1 nephritis and Heymann nephritis. FcRn may play a role in the development of immune-induced glomerulonephritis.

Animals ; Glomerulonephritis, IGA ; metabolism ; pathology ; Glomerulonephritis, Membranous ; metabolism ; pathology ; Glomerulosclerosis, Focal Segmental ; metabolism ; pathology ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Humans ; Laser Capture Microdissection ; Lupus Nephritis ; metabolism ; pathology ; Male ; Nephritis ; genetics ; immunology ; metabolism ; pathology ; Nephrosis, Lipoid ; metabolism ; pathology ; Podocytes ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Fc ; genetics ; metabolism ; Thy-1 Antigens ; immunology ; metabolism ; Up-Regulation

PURPOSE: This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs). MATERIALS AND METHODS: Aliquots (10 g) of the lipoaspirates were stored at 4degrees C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed. RESULTS: When the lipoaspirates were stored at 4degrees C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1x1014 fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells. CONCLUSION: ASCs isolated from lipoaspirates and stored for 24 hours at 4degrees C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4degrees C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.
5'-Nucleotidase/metabolism ; Adipose Tissue/*cytology ; Adult ; Antigens, CD/metabolism ; Antigens, CD44/metabolism ; Antigens, Thy-1/metabolism ; Cell Differentiation/physiology ; Cells, Cultured ; Female ; Humans ; Immunoblotting ; Immunohistochemistry ; Immunophenotyping ; Mesenchymal Stem Cells/metabolism ; Muscle Development/genetics/physiology ; Osteogenesis/genetics/physiology ; Receptors, Cell Surface/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells/*cytology/metabolism ; Young Adult

AC133 Antigen ; Adult ; Aged ; Antigens, CD ; metabolism ; Carcinoma, Hepatocellular ; diagnosis ; metabolism ; pathology ; Female ; Glycoproteins ; metabolism ; Humans ; Liver Neoplasms ; diagnosis ; metabolism ; pathology ; Male ; Middle Aged ; Peptides ; metabolism ; Prognosis ; Thy-1 Antigens ; metabolism

OBJECTIVETo investigate the role of transcription factor special AT-rich binding protein 2 (SATB2) in the osteoblasts differentiation of bone marrow stromal cells (BMSC) in vitro.

METHODSRats bone marrow stromal cells were isolated by Percoll sedimentation and the cells were placed and allowed to attach for three times. After passages, expression plasmid pBABE-hygro-satb2 was constructed, then transfected into BMSC. BMSCs were inoculated in conditioned medium and osteogenic factors were detected by western blotting and reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe morphological observation of BMSC showed either spindle or polygonal pattern. The cellular phenotypic marker of the third passage was CD29 positive and CD34 negative. The growth curve possessed "S" pattern. The intensity of calfilication in BMSC was higher in SATB2 transfection group (IA value 125974 ± 241) than that in the control groups (IA value 178486 ± 406). Moreover, cell migration rate increased in SATB2 transfection group [width of scratch (0.72 ± 0.01) mm] compared with control group [width of scratch (0.83 ± 0.03) mm]. In addition, the mRNA expression of osteogenic factors runt-related transcription factor 2, Osterix, activating transcription factor 4, integrin-binding sialoprotein were upregulated.

CONCLUSIONSCells cultured with this method have general biological characteristics and osteogenic differentiation potential in vitro. SATB2 can promote osteoblasts differentiation of BMSC.

Activating Transcription Factor 4 ; metabolism ; Animals ; Bone Marrow Cells ; metabolism ; pathology ; Cell Differentiation ; Cell Movement ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Integrin-Binding Sialoprotein ; metabolism ; Male ; Matrix Attachment Region Binding Proteins ; genetics ; metabolism ; Osteoblasts ; cytology ; Osteogenesis ; Plasmids ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stromal Cells ; metabolism ; pathology ; Thy-1 Antigens ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection

This study was designed to construct THY1 eukaryotic expression plasmid and assess its effects on epithelial ovarian cancer cell line SKOV3. The gene fragment coding for THY1 was inserted into pcDNA3.1(+) for constructing the recombinant plasmid pcDNA3.1(+)-THY1. The eukaryotic expression plasmid was analyzed by PCR, restriction endonucleases digestion and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-THY1 was transfected into SKOV3 cells by liposome protocol. The experimental cells were classified into three groups: SKOV3-THY1, SKOV3-Null and SKOV3. The pcDNA3.1(+)-THY1 has been transfected into SKOV3 cells by RT-PCR and immunohistochemistry. The cell inhibitory rate of SKOV3-THY1 (56.6% at the fifth day) was higher than that of SKOV3-Null (12.5%), there was significant difference between them (P<0.05). The ratios of G1 phase of SKOV3 cells after transfection were increased and the ratios of S phase were decreased significantly. There was significant difference between SKOV3-THY1 and SKOV3-Null or SKOV3 (P<0.05), but there was no significant difference between SKOV3-Null and SKOV3 (P>0.05). We have constructed the recombinant plasmid pcDNA3.1(+)-THY1 sucessfully. THY1 transfection can inhibit the growth of SKOV3 cells in vitro.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cloning, Molecular ; Female ; Genetic Vectors ; genetics ; Humans ; Ovarian Neoplasms ; pathology ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Thy-1 Antigens ; biosynthesis ; genetics ; Transfection

OBJECTIVETo isolate and characterize human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs).

METHODSThe synovial membrane tissues were obtained from 4 RA patients, 1 chondroma patient and 1 healthy subject and FLS were isolated by means of tissue culture. The cell morphology was observed by phase-contrast microscope and the cell surface markers were detected by flow cytometry.

RESULTSThe FLSs were successfully cultured from the synovial membrane tissues with good cell homogeneity after the third passage. The FLSs of the 3rd to 7th passages were stable and proliferated actively, followed by slow proliferation and aging since the 8th passage. Flow cytometry showed that the 4th-passage FLSs from the RA patients contained 99.04% CD90(+) cells, 2.73% CD3(+) cells, 0.29% CD3(-)CD19(+) cells, 2.81% CD3(-)CD16(+)CD56(+) cells, 5.89% CD14(+) cells, and 54.17% CD55(+) cells. The presence of interleukin-1 receptor type I (IL-1RI, 158.63-/+20.32 pg/ml) and IL-1beta (4.67-/+0.82 pg/ml) were detected in the cell culture supernatant of the 4th-passage FLSs from the RA patients by enzyme-linked immunosorbent assay ELISA.

CONCLUSIONFLSs from RA patients can be effectively culture by means of tissue culture, and the cultured FLSs show high expressions of CD90, IL-1RI and IL-1beta.

Adult ; Aged ; Arthritis, Rheumatoid ; pathology ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Female ; Fibroblasts ; pathology ; Humans ; Interleukin-1beta ; metabolism ; Male ; Middle Aged ; Receptors, Interleukin-1 Type I ; metabolism ; Synovial Membrane ; cytology ; pathology ; Thy-1 Antigens ; metabolism