ABSTRACT
BACKGROUND: The mostly accepted standpoints regarding adult stem cell plasticity are that adult stem cells existing in various organs contain primitive pluripotent stem cells. However, the origin, identification methods and biological characteristics of pluripotent stem cells remain unclear. OBJECTIVE: To investigate if a kind of pluripotent stem cells with more primitive character can be isolated from adult tissues. DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Center of Tissue Engineering, Basic Medical Department of Peking Union Medical College from March 2007 to January 2008. MATERIALS: Pregnant BALB/C rats on gestation day of 12.5-14.5 d were adopt in the study. METHODS: The fetal perisome samples were attained from pregnant BALB/C rats under sterile conditions, and made into cell suspension by trypsin digestion method. Immunomagnetic beads were used to sort Sca-1+CD117-Lin-cells. When 70%-80% cells were mixed, the cells were subcultured, and the 5 passage cells were taken in the experiment. MAIN OUTCOME MEASURES: Microstructure, ultrastructure, growth characteristics, immunophenotype and embryonic stem cell associated antigen were analyzed by inverted phase contrast microscope, scanning electron microscope, flow cytometry and RT-PCR respectively. Histochemical and immunofluorescence staining were used to investigate the potentiality of the Sca-1+CD117-Lin-cells differentiated into trilaminar germ disc. RESULTS: Sca-1+CD117-Lin-cells adhering to the wall with spindle or short fusiform shapes, which could amplification fast and stablely to 50 passages. Cells were found logarithmic growth on days 3-7, after that cell growth reached platform stage. There were about 32 h for doubling time, 90.43% cells were at the G0/G1 phase, and 9.57% at the G2/M+S phase. The cells expressed high levels of Sca-1, no levels of CD117, CD14, CD19, CD31, CD34, CD45 and Flk-1, and moderate levels of CD44. The phenotype of human BMSCs was stabilization. Embryonic stem cell associated antigen Sox2, Oct-4 or Nanog were showed positive expression. RT-PCR indicated that the cells were able to differentiate into osteoblasts, adipocytes, chondrocyte, hepatocyte-like cells and neuroglia cells in vitro. CONCLUSION: The Sca-1+CD117-Lin-cells isolated from adult tissue are primitive multiple potential adult stem cells, which can differentiate into trilaminar germ disc.
ABSTRACT
Objective To study the effect of Sca1+ cells on the HSCs`(hemopoietic stem cells) differentiation to dendritic cells(DCs),and identify the morphology,function and surface markers of the cells.Methods CD117+ HSCs were isolated and purified from the bone marrow of healthy Balb/c mice by magnetic affinity cell sorting.After cell expansion by treatment with the support of Sca1+ cells,the HSCs were induced for directional differentiation into DC-like cells.Studied the surface markers by FACS and function via LSCM and animal experiments. Results After 10 days of culture,we demonstrated that Sca1+ cells induced HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia.They had phagocytotic activity,and suppressed the GVHD(graft versus host disease) reaction.Conclusion HSCs can differente into dendritic cells with the support of Sca1+ cells.
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the feasibility and characteristics of human engraftment in HLA disparate cord blood transplantation.</p><p><b>METHODS</b>Two human HLA-haploidentical or HLA-mismatched cord blood units were transplanted into sublethally irradiated severe combined immunodeficiency (SCID) mice. The characteristics of engraftment, hematopoietic and immunological reconstitution between the two groups were compared.</p><p><b>RESULTS</b>Two mixed cord blood units can engraft in SCID mice with donor-recipient chimerism and reconstitute hematopoiesis and immunological functions. No unfavorable factors had been observed. Only one of the two cord blood units which had higher colony forming ability in vitro could engraft in most SCID mice as shown by HLA-DQB(1) gene detection. Two HLA-haploidentical cord blood units were simultaneously engrafted in 3 SCID mice.</p><p><b>CONCLUSION</b>Double HLA-haploidentical or HLA-mismatched cord blood can engraft in SCID mice and reconstitute hematopoietic and immunological functions. HLA disparity has no significant effect on survival and engrafting rate. However, in less HLA disparity group, two cord blood units were prone to engraft simultaneously.</p>
Subject(s)
Animals , Female , Humans , Mice , Antigens, CD , Allergy and Immunology , Cord Blood Stem Cell Transplantation , Methods , Disease Models, Animal , Fetal Blood , Allergy and Immunology , Metabolism , Flow Cytometry , HLA Antigens , Genetics , Allergy and Immunology , Hematopoiesis , Mice, SCID , Random Allocation , Severe Combined Immunodeficiency , Allergy and Immunology , General Surgery , Survival Analysis , Transplantation, HeterologousABSTRACT
AIM: To study the effect of vascular endothelial growth factor(VEGF) on hematopoietic differentiation from mouse embryonic stem cells(ESC) in vitro.METHODS: ES-D3 was allowed to grow on mouse fetal fibroblast feeder layer,and then was developed into embryoid bodies(EB).EB cells were transferred into medium supplemented with different concentration of VEGF and VEGF+SCF for 1 week.Six groups,including.VEGF 5 ?g/L,VEGF 10 ?g/L,VEGF 20 ?g/L, VEGF 5 ?g/L+SCF,VEGF 10 ?g/L+SCF and VEGF 20 ?g/L+SCF,were designed.The group of spontaneous differentiation without cytokine(s) was used as control.Hematopoietic transcription factor GATA-2 and early hematopoietic differentiation genes(c-kit and ?-H1) were detected by RT-PCR.The content of CD34~+ cells in each group were measured by flow cytometry.The cells derived from ESC were incubated in semisolid methycellulose cultures.The numbers of total colony-forming units in culture(CFU-C) were counted by reverse microscope.RESULTS: ES-D3 grew and formed EB at day 4.VEGF had a stimulatory effect as a single factor on the expression of genes associated with early hematopoietic differentiation(GATA-2,c-kit and ?-H1),the generation of CD34~+ cells and CFU-C in EB.The effects of VEGF+SCF were the most potent in the experimental groups according to the percent of CD34~+ cells and the numbers of hematopoietic colonies.The most highest inducing efficacy was achieved in VEGF 20 ?g/L or VEGF 10 ?g/L combined with SCF.CONCLUSION: VEGF promotes the differentiation of ESC into hematopoietic cells in vitro.The strongest effect was achieved when VEGF was combined with SCF.