| Part Ⅰ Isolation, culture and identification of cord-blood derived endothelial progenitor cells(CB-EPCs)Aims. To explore the culture method for CB- EPCs, and establish one stable culture system in our laboratory.Methods. After isolation of mononuclear cells(MNCs) from CB by density- gradient centrifugation method, the cells were seeded in culture flask coated with fibronectin and then cultured for 72 hours(h) with endothelial growth medium-2(EGM-2), which contained recombinant human fibroblast growth factor-β(rh FGF-β), recombinant human epidermal growth factor(rh EGF), R3- insulin- like growth factor(IGF)-1, vascular endothelial growth factor(VEGF), ascorbic acid and GA-100) and 10%FBS. After 72 h, non-adherent cells were removed while the adherent ones continued to be cultured. When the cultured cells manifested typical EPC morphology and merged to 80%, the cells were then passaged. Flowcytometry was used to analyze the surface antigen of the cultured cells. And the antigens included CD34, CD133, CD45, KDR, CD31 and CD14. By using CCK8 reagent, the proliferation ability of EPC was measured, with mature endothelila cells human umbilical veins endothelial cells, HUVECs as control. The tube formation ability of EPC was analyzed by using Matrigel reagent and the migration ability by Transwell chamber. Real time quantitative polymerase chain reaction(RT-q PCR) was applied to analyze relative endothelial genes to further identify EPC characteristics.Results. Under culture condition in which non-adherent cells were removed after 72 h with special medium, typical morphology of EPC was emerged at day 21-day 28. These spindled cells merged as colonies first and the colonies merged then with fast proliferation ability. The cultured cells could be passaged to 10. Flowcytometry analysis revealed that both C B-EPCs and HUVECs expressed the endothelial markers such as KDR(EPCs vs. HUVECs, 34.21%±6.44% vs. 30.78%±5.60%, P=0.524) and CD31(EPCs vs. HUVECs, 98.82%±1.33% vs. 97.90%±1.50%, P=0.472), and CB-EPCs manifested a higher level of progenitor cell markers including CD34(EPCs vs. HUVECs, 45.66%±6.81% vs. 26.89%±4.91%, P=0.018) and CD133(EPCs, 26.12%±4.56%), but very weakly positive for CD45(EPCs vs. HUVECs, 2.32%±1.56% vs. 0.92%±0.77%, P=0.236) and CD14(EPCs vs. HUVECs, 3.44%±1.27% vs. 1.29%±0.54%, P=0.054), indicating that the EPCs were not of hematopoietic origin and nearly not contaminated with other cell types. The EPC showed considerably enhanced proliferative potential when compared with HUVEC, and the EPC used within 5 passages in our experiment had comparatively high proliferation potential. The number of tubes formed by EPC(P1, 28.67 ± 4.51; P3, 29.33 ± 4.51; P5, 31.33 ± 4.04, were observed at 40 x) was less than HUVEC(71.67 ± 7.64, 40×). The tubes formed by different passages of EPC were similar(P = 0.749). After a brief incubation, more EPC(P1, 208.67 ± 13.05; P3, 204.33 ± 16.17; P5, 203.00 ± 16.37, were observed at 100 × under the chamber than HUVEC(71.67 ± 3.79), without any difference among EPC passages(P = 0.925), indicating a more rapid migration capacity of EPC. The transcriptional levels of VWF and VEGF, were increased by 4.49 ± 0.50(P = 0.000) and 1.76 ± 0.12(P = 0.008) fold in EPC than HUVEC respectively. EPC expressed higher TIE-1 gene(2.08 ± 0.28 fold, P = 0.021), TIE-2(2.25 ± 0.26 fold, P = 0.014) and SELE gene(7.34 ± 0.61 fold, P = 0.000) when compared with HUVECs.Conclusions. By methods that isolating MNCs from CB, and plating MNCs on fibronectin coated culture flask for 72 h with special medium, then removing non-adherent cells, we can harvest CB-EPC at day 21-day 28 after culture. Phenotypic functional analysis had ensured the cultured cells were CB-EPCs orgins. We have made important basis for further study.Part Ⅱ Cord blood derived endothelial progenitor cells(CB-EPCs) acted as stroma to expand hematopoietic stem/ progenitor cells(HSPCs) ex vivoAims. Hematopoietic stem cell transplantation(HSCT) of CB has often been limited by the scarcity of stem cells. Therefore, the number of CB-HSPCs should be increased while maintaining the stem cell characteristics. We here to explore the ability of CB-EPCs to support HSPCs expansion ex vivo and relevant mechanism.Methods. CB-EPCs were isolated and cultured routinely. HSPCs cells were originated from different donors with EPCs. CD34+ HSPCs were positively sorted by magnetic beads and identified with flowcytometry. EPCs and HSPCs were co-cultured by direct contact and non-direct contact culture methods separately, with HSPCs cultured alone as control. And after 7 days of culture, the total number of expanded cells, the number of C D34+ cells, more primitive CD34+CD38- cells, lineage progenitors CD34+CD33+ cells, CD34+C D19+ cells and CD34+CD61+ cells were measured. Besides, cell cycle analysis were done by flowcytometry. And the functional analysis of expanded HSPCs were evaluated. Hematopoietic reconstitution ability in vivo were carried out in a xenograft model with non-obese diabetic/severe combined immunodeficient mice(NOD/SCID). And the survival, peripheral blood recovery and implantation of mice were analyzed.Results. After seven days of incubation, the cells cultured alone with cytokines had a 3.76 ± 0.60 fold expansion. The contact of HSPCs on CB-EPCs supported HSPCs a 6.75±0.84 fold expansion(P = 0.003). The noncontact co-culture of HSPCs contributed to a higher(7.56±0.80 fold) increase in total cell numbers compared with HSPCs cultured alone(P = 0.001), and had an similar cell expansion fold to direct contact co-culture system(P = 0.239). C D34+ cells also showed significant increase(4.06 ± 0.43) under non-contact conditions compared with cytokine-cultured cells(P = 0.025). Contact co-culture of HSPCs with CB-EPCs owned 156.17 ± 21.32 fold expansion of the much earlier subtype of CD34+C D38- cells, which was higher than that of the HSPCs cultured alone(with 79.12 ± 19.77 fold increase, P = 0.010) and with non-contact co-culture(with 95.43 ± 33.00 fold increase, P = 0.026). Under contact co-culture condition, the CD34+CD33+, CD34+CD19+ and CD34+CD61+ populations had a much higher expansion than single culture or non-contact co-culture condition. HSPCs cultured alone showed a decrease of cells in G0/G1 phase(60.67% ± 1.68%, P = 0.000), an increase of cells in S phase(31.40% ± 3.50%, P = 0.000), and comparable cells in G2/M phase(5.94% ± 0.17%, P = 0.120) compared with HSPCs on day 0. And EPCs contact co-culture induced 59.77% ± 1.94%(P = 0.000) of HSPCs into G0/G1 phase, 35.17% ± 2.75% into S phase(P = 0.000) and 5.62% ± 0.39% into G2/M phase(P = 0.851). The clonogenic potential of the HSPCs under contact co-culture was demonstrated much higher total CFU numbers and higher number of CFU-GM,BFU-E and CFU-E than those single cultured. In NOD/SCID system, mice transplanted with progeny of HSPCs after contact co-culture showed a similar recovery trend of blood cell count as those with freshly isolated HSPCs, while mice transplanted with single culture HSPCs showed mild recovery around 3 to 4 weeks but declined and died soon within 5 weeks. There was no significant difference in survival between the mice transplanted with progeny of HSPCs after contact co-culture and freshly isolated HSPCs and both had full observation period. The above two transplantation groups were engrafted with human HSPCs, with 16.0% ± 14.3% and 13.3% ± 11.0% engraftment respectively.(P = 0.750). The transcription of hematopoietic related cytokines genes IL-6(4.24 ± 1.42, P = 0.017), ANGPT1(7.75 ± 0.90, P = 0.000) and CXCL12(8.25 ± 2.03, P = 0.025) were elevated in CB-EPCs compared with MSCs. And Wnt5 A, the key ligand in Wnt signaling pathway, exhibited an 11.76 ± 2.11 fold increase in EPCs compared with MSCs, and also higher in the co-cultured HSPCs, with a 3.49 ± 0.54 fold increase(P = 0.001) than cells before co-culture.Conclusions. EPCs are capable of expanding the HSPCs ex vivo and induce primitive cells expansion. They could produce various growth factors and signa ling molecules and activate some key signaling pathway. Pluripotent HSPCs need to interact directly with EPCs to maintain their stem-cell characteristics. The improved co-culture method offers a efficient and selective way to expand CB-HSPCs for transplantation clinically.Part Ⅲ Primary study of cord blood derived endothelial progenitor cells(CB-EPCs) combined with hematopoietic stem cell transplantationAims. To explore whether CB-EPCs could repair the endothelial injury during HSCT, promote hematopoietic recovery and red uce graft-versus- host disease(GVHD).Methods. To establish CB-EPCs combined with HSCT animal model first, with mice transplanted with HSCs alone as control. The survival time, peripheral blood recovery GVHD syndrome were observed after transplantation. GVHD scoring and pathological identification were necessary. Immnuohistochemistry with CD31 were used to evaluated micro-blood vessels recovery after EPC co-transplantation, with mice transplanted only with HSCs as control. EPCs transfected with EGFP and to be detected after transplantation, to find out whether EPCs directly homing to bone marrows and repairing the injured endothelial cells. The immunogenicity of EPCs were analyzed by detecting the HLA antigen on surface and mixed lymphocyte reaction, with MSCs as control.Results. The median survival time for mice(n=10) with single HSCT was 51 days, with a 60% long-term survival rate. While the median survival time for mice(n=10) co-transplanted with CB-EPCs was 54 days, with a 70% long-term survival rate. Though there was a higher survival time for co-transplanted mice than single-transplanted mice, no significance was detected(P=0.314). Besides, the two transplantation groups both had recovery trends of peripheral blood, and the co-transplantation group had higher blood cell counts at every detection point. The bone morrow chimerism of human CD45 cells in single transplantation group was 13.27%±6.36%, while that of co-transplantation group was 18.21%±10.57%. However, there was no detected significance(P=0.308). On the other hand, the spleen chimerism in single transplantation group was 4.91%±7.56%, and in co-transplantation group it was 5.14%±2.97%, with no significance detected(P=0.840). There were 3 mice in single transplantation group, with scoring of 2, 2 and 6 respectively. By contrast, there were only 2 mice in co-transplantation group, with scoring of 2 and 1. Pathological analysis confirmed the GVHD injury in bone marrow and intestinal. Mice with co-transplantation had obvious and continuous CD31 stain, which suggested good recovery of micro-blood vessels. But those with single transplantation recovered less in bone marrow micro-blood vessels. After transplanted with EGFP-EPCs for 24 hours, there were detective positive cells in bone marrow with a mean percentage of 2.3%, which lasted to 72 hours and 7 days but became lesser. The immunogenicity of EPC was low, and had a weaker HLA antigen express than MSCs, with HLA-ABC(27.97%±3.60% vs. 44.46%±7.18%, P=0.024) and HLA-DR(6.37%±2.73% vs. 14.77%±1.96%, P=0.012). And no excessive lymphocyte proliferation in mixed lymphocyte reactions.Conclusions. Our results suggested that CB- EPCs owned the potential to promote hematopoietic reconstitution, reduce GVHD and prolong survival time. However, the results were limited by sample number and need to be further elucidated. And repair of bone marrow micro-blood vessels might be one of the mechanism. |
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