ABSTRACT
BACKGROUND:Stem cels can induce immune tolerance, prolong graft survival time and reduce rejection in organ transplantation, which have become a hot research. OBJECTIVE:To induce immune tolerance to alogenic kidney transplantation with amniotic fluid stem cels in recipient rats and to explore the mechanism underlying immune tolerance. METHODS: Amniotic fluid stem cels were isolated from Wistar rats. Two inbred male rat strains, Wistar rats and Sprague-Dawley rats, were selected as donors and recipients of kidney transplantation. The rat models of renal orthotopic transplantation were divided into the folowing four groups: a sham-operated group (n=10, Sprague-Dawley rats); an isograft group (n=10, Sprague-Dawley to Sprague-Dawley rats); a control group (n=10, Wistar to Sprague-Dawley rats, treated with 1 mL saline); and an experimental group (n=10, Wistar to Sprague-Dawley rats, treated with 1 mL of 3×106/L amniotic fluid stem cels). Serum levels of creatinine, urea nitrogen, interleukin-2, interferon-γ, parameters of oxidative stress were detected at 5 days after operation. Flow cytometry was employed to determine the percentage of CD4+ and CD8+ lymphocytes in the peripheral blood. Kidney transplants were observed pathologicaly. RESULTS AND CONCLUSION:Compared with the control group, the levels of creatinine, urea nitrogen, interleukin-2, interferon-γ, parameters of oxidative stress and proteinuria were lower in the experimental group (P < 0.05). Percentages of CD4+, CD8+ and CD4+/CD8+ ratio were also significantly lower in the experimental group than the control group. However, the rate of cretinemia clearance in the experimental group was significantly higher than that in the control group (P < 0.05). Furthermore, the degree of kidney injury in the experimental group was significantly lower than that in the control group. Our findings demonstrate that the amniotic fluid stem cel transplantation can induce immune tolerance, extenuate oxidative stress, attenuate pathological damage to the kidney transplant and preserve kidney function from acute rejection in rats undergoing kidney transplantation.
ABSTRACT
BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.