ABSTRACT
ObjectiveTo investigate the potential of human amnion-derived mesenchymal stem cells to differentiate into insulin secreting cells in vitro.MethodsThe hAD-MSCs were isolated from human amnion by trypsin-collagenase digestion.The phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.The 3rd generation cells were inoculated at density of 2.5 × 106 unit/ml or 5 × 105 unit/ml in 6 well plates or preset coverslip 24 well plates.Induced groups were treated in serum-free HG-DMEM with 10 mmol/L nicotinamide and N2 supplement.The cells in the non-induced groups were incubated in LG-DMEM supplemented with 10％ fetal bovine serum.At days 7,14 and 21 after induction,insulin and β2 microglobulin was determined by immunocytochemical stain,the content of insulin in the culture supernatant was assayed by radioimmunoassay,and insulin mRNA and PDX-1 mRNA were detected by reverse transcriptase-polymerase chain reaction.Results( 1 ) The hAD-MSCs highly expressed CD29,CD44,CD73,CD166,and vimentin.( 2 ) At 7,14,and 21 days,the percentages of insulin-positive cells in hAD-MSCs induced groups were 74.67％ ± 1.53％,75.00％:1.00％,and 74.33％ ±1.53％,respectively.Contents of insulin in the supematant of hAD-MSCs induced groups were ( 331.62 ± 1.76 ),( 330.50 ± 1.22 ),and ( 331.65 ± 0.48 ) μIU/ml,respectively,but non-induced groups were negative.(3) PDX-1 mRNA and PDX-1 protein were expressed before and after the induction of hADMSCs,but insulin mRNA was expressed only in the induced groups.( 4 ) Both hAD-MSCs induced groups and non-induced groups expressed β2 microglobulin ( all P ＞ 0.05 ).ConclusionThe hAD-MSCs have a potential of differentiating into ISCs and thus may become a new cell source of therapy for type 1 diabetes.
ABSTRACT
BACKGROUND: As is well known that hematopoietic stem and progenitor cells (HSPCs) contain in bone marrow, peripheral blood, and cord blood. Recent studies found that human placenta tissue (PT) also exists in HSPCs. But so far the property and differentiation capacity of human PT-HSPCs is not yet known. Furthermore the composition of lymphocyte subpopulations and immunogenicity regarded to human PT-HSPCs are also unclear.OBJECTIVE: To verify whether there are more HSPCs in human PT than those in human umbilical cord blood (UCB), to investigate their capacities of proliferation and differentiation, and to analyze the phenotypes of lymphocyte subpopulations in human PT.DESIGN, TIME AND SETTING: Open eXperiments were performed at the Key Laboratory of Cell Engineering of Guizhou Provinee from January 2004 to December 2006.SETTING: Key Laboratory of Cell Engineering of Guizhou Province, the Affiliated Hospital of Zunyi Medical College.MATERIALS: Twelve human placenta and UCB samples through cesarean delivery were collected aseptically with the informed consents of parturients derived from Maternity Department of the Affiliated Hospital of Zunyi Medical College. The main reagents were detailed as follows: lymphocyte subpopulations analysis reagents Simultest IMK-lymphocyte Kit, CD34 absolute counting reagents Kit (Becton Dickinson); CD34 Multisort Kit, FITC conjugated CD38 monoclonal antibody, anti-FITC microbeads and MS/LS mini MACS segregating, columns (Miltenyi Biotec).METHODS: UCB samples were 1:1 diluted with RPMI-1640 containing 0.1 volume fraction of fetal bovine serum and the mononuclear cells (MNCs) were isolated on Ficoll-Histopaque by centrifugation for 30 minutes. The MNCs at the interface were collected and washed with PBS. Single cells suspension liquid of human PT was prepared by mechanical method combined with 0.25g/L collagenase digestion. After that, the placenta samples underwent the same protocol as used in UCB to isolate MNCs. The percentage of CD34+CD38-, CD34+CD38+ HSPCs and the phenotype of lymphocyte subpopulations derived from human PT-MNCs were analyzed by flow cytometry (FCM). CD34+CD38-, CD34+CD38- cell subsets isolated by magnetic-activated cell sorting (MACS) from human PT were used to carry out colony-forming culture including granulocyte/macrophage colony-forming unit (CFU-GM), burst forming unit-erythroid (BFU-E) and mixed colony-forming unit (CFU-Mix) in order to assess their capacities of hematopoietic progenitor cells' proliferation and differentiation. In parallel, UCB samples underwent the same protocols for comparison.MAIN 0UTCOME MEASURES: Percent compositions of CD34+ HSPCs, hematopoietic progenitors' lineage colony-forming capacities of CD34+ HSPCs, phenotypes and compositions of lymphocyte subpopulations both in PT and UCB.RESULTS: The percentage of CD34+ cells contained in human PT was 8.8 times higher than that of in UCB (P<0.01). The total number of lymphocytes, T cells (CD3+CD2+), B cells (CD19+), Th (CD3+CD4+) and Th/Ts ratio were apparently lower in human placenta, while the number of CD8+CD28- T suppressor cells were higher compared to UCB samples (P<0.01). Among PT, CFU-GM, BFU-E and CFU-Mix frequencies of CD34+CD38- cells subset were much higher than that of CD34+CD38- (P<0.01). Within the same phenotype of cell subsets, however, the number of each colony-forming unit was similar between PT and UCB (P 0.05).CONCLUSION: Human PT is richer in CD34+CD38-, CD34+CD38+ HSPCs and both of them have the abilities of proliferating and differentiating into CFU-GM, BFU-E and CFU-Mix. Considering that human PT have a lower lymphocyte subpopulations and higher Ts cells, human PT might be a alternative and suitable source of HSPCs for clinical transplantation.
ABSTRACT
AIM:To investigate the activity of NF-?B in peripheral blood mononuclear cells (PBMCs) from patients with Graves disease (GD) and the significance in immunopathogenesis of GD.METHODS:Peripheral blood was collected from 22 untreated GD,20 treated GD with tapazole more than 1 year,and 25 healthy volunteers. PBMCs were isolated from the blood by histopaque-1077 density-gradient centrifugation. The activity of NF-?B in PBMCs was analyzed using gel electrophoretic mobility shift assay (EMSA). The contents of IL-1?,IL-6 and TNF-? were tested by radioimmunoassay.RESULTS:The activity of NF-?B in PBMCs of untreated GD group was increased remarkably,compared with that in the treated group and control (P