Properties of hematopoietic stem/progenitor cells derived from human placenta tissues / 中国组织工程研究

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 11 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.