ABSTRACT
BACKGROUND: The proliferation of peripheral blood stem cells among peripheral blood mononuclear cells (PBMCs) invitro remains unclear. There is no optimal marker for tracing PBMCs transplanted in vivo.OBJECTIVE: To observe the degree of PBMC proliferation in stem cell medium by EdU labeling and to explore thefeasibility of EdU-labeled peripheral blood stem cells.METHODS: New Zealand rabbit PBMCs were isolated and cultured for 1 to 5 days in stem cell medium supplementedwith EdU. The cells were observed and counted at 0, 1, 2, 3, 4 and 5 days in culture. The cells were harvested at eachtime point and stained with EdU fluorescent reagents. Then, confocal microscopy and flow cytometry were used to detectEdU-labeled cells.RESULTS AND CONCLUSION: (1) Freshly isolated rabbit PBMCs were rounded and showed clear outline. After 1 dayculture, most of the cells were suspended in the medium, spherical or round. There were also a few cell clusters andadherent cells scattered in a triangle or polygon shape; after 2 days culture, more cell debris were observed, and mostcells were round; when cultured for 3-5 days, increased cell debris, smaller cell mass and decreased cell densitysignificantly were observed. (2) With the prolongation of culture time, the cell count decreased gradually. (3) Whencultured for 1 day, EdU labeled cells in red were scattered. The number of cells marked with EdU red label increasedsignificantly at day 2 and remained unchanged after 3 days of culture. At 5 days of culture, the number of red cellsmarkedly decreased; the highest positive rate of EdU-labeled cells was (2.38±0.10)% at 2 days after culture. To conclude,these results showed that the proportion of proliferating cells in rabbit PBMCs was very low. EdU is capable of labelingproliferative cells among PBMCs.
ABSTRACT
BACKGROUND: Previous research has demonstrated that dermal tissue has mesenchymal stem cells, which have a possibility of autologous transplantation. If the mesenchymal stem cells derived from the skin differentiate into lymphocytes under a certain condition, the immune system disease can be solved generally.OBJECTIVE: To investigate the possibility of differentiation of human skin-derived mesenchymal stem cells into lymphocytes. METHODS: Surface marker expression was detected in the 14th passage human skin-derived mesenchymal stem cells using flow cytometry. Transdifferentiation medium of human skin-derived mesenchymal stem cells consisted of human lymphocyte supernatant and fresh human skin-derived mesenchymal stem cells based on the ratio of 7:3. Inverted microscope was employed to observe morphological changes, and flow cytometry was used to detect surface marker expression in the lymphocytes at 1-8 days after induction. Self-marker expression of human skin-derived mesenchymal stem cells was then detected at 3,6, and 9 days after induction.RESULTS AND CONCLUSION: Human skin-derived mesenchymal stem cells stably expressed self-specific marker CD73, Vimentin and so on, but did not express specific markers of hematopoietic system, I.e., CD34, CD45 and so on, lowly expressed HLA-I, but did not express HLA-DR at all. At 3 days after induction, the cell volume significantly increased, cell proliferation rate was significantly lower than before induction, and a lot of cystic-like particles with strong refraction were observed in or between cells. The CD45 lymphocyte expression was not significantly changed, but CD3, CD19, CD16, CD4, and CD8 expression rates of human skin-derived mesenchymal stem cells were linearly increased at 1-4 days after induction and stabilized at 5-8 days after induction. In addition, CD37, CD34, Vimentin, and HLA-DR expressions were not changed at 3, 6, and 9 days after induction, but HLA-I expression rate was gradually increased with the prolongation time of induction. This suggested that human skin-derived mesenchymal stem cells can differentiate into lymphocyte and potentially participate in repairing immune system injury.