Immunophenotypic Analysis of Umbilical Cord Blood Stem Cells

ABSTRACT

PURPOSE: Since umbilical cord blood (UCB), which used to be discarded, was found to be a source of enriched hematopoietic stem and progenitor cells, basic research to elucidate characteristics of UCB hematopoietic stem cells (HSCs) and its clinical application to bedside transplantation have been attempted. Moreover, stem cell transplantation (SCT) has expanded its role, not only in hematopoietic reconstitution, but in cancer therapy, stem cell expansion and gene therapy. This study was aimed to clarify the characteristics of UCB HSC comparing differences between term and preterm babies in term of 1) hematologic parameters, 2) immunophenotypic characteristics studied by flow cytometer utilizing CD34 and several other monoclonal antibodies (MoAb), and 3) hematopoietic capacity by clonogenic assays. METHODS: UCB was obtained from 18 term babies and 13 preterms after informed consents. Samples were initially tested for complete blood counts. Immunophenotypic characteristics were studied in 11 cases (preterm, 4; term, 7) by two laser FACscan plus (Becton Dickinson) with FITC-conjugated MoAb to CD3, CD4, CD5, CD8, CD10, CD16+ 56, CD19, CD33, CD34, CD38, CD71, Thy-1, and HLA-DR. Clonogenic assays were performed by methylcellulose method. RESULTS: The mean hematologic parameters for all groups were white blood cell, 12.7x103/microliter; hemoglobin 14.8g/dL; platelets, 230x103/microliter. The parameters for preterms and terms were as follows white blood cell, 10.6x103/microliter vs 13.9x103/microliter; hemoglobin 13.5g/dL vs. 15.4g/dL; platelets, 188x103/microliter vs 254x103/microliter; mononuclear cells 5.1x103/microliter vs 6.4x103/microliter, respectively. Parameters other than hemoglobin and platelet counts were not significantly different between the two groups. The colony-forming units granulocyte-macrophage (CFU-GM) count and count for all colonies identified on day 14 were 10,888+/-11,257.3/mL and 16,504+/-16,531.6/mL, respectively. However, there was no significant differences in clonogenic assays between the term and preterm groups. The percentage of CD34+ cells in mononuclear cells was 1.5+/-1.5%, with 1.0+/-0.2% for preterms and 1.8+/-1.9% for terms. The number of CD34+ cells was 5.5+/-4.1x104/mL, with 3.8+/-2.0x104/mL for preterms and 6.5+/-4.7x104/mL, respectively. These findings suggested that the percentage and number for CD34 cells and the number of CFUs be higher in term babies than in preterms, but the differences failed to meet statistical significances. As T cell markers, CD3 (pan-T cell) and CD5 (early developmental T cell) were positive in 28.5% and 32.8%, respectively. The CD4 CD8 ratio for all was 2.2+/-0.5, with 2.3+/-0.3 for preterms and 2.1+/-0.6 for terms, respectively, tending to decrease with gestational age with transient increase when approaching to the term. CD10 and CD19 expression as markers for B cell-associated antigens were 1.8+/-1.6% and 6.5+/-4.6%, respectively. Myeloid marker CD33 was positive in 2.24%, while CD71 (transferrin receptor) in 43.7%. Thy-1 was 30.0% with peak of 63.4% at 32th gestational week. As a subpopulation study among HSCs, CD34+CD38- cells were 2.1+/-1.5%, CD34+ HLA-DR+ was present in 85.3+/-3.1%, while CD34+CD19+ cells were 1.7+/-1.6%. CONCLUSION: These results suggested that T cells in UCB were immature, that the number of CD8+ cells which are known to be implicated in graft-versus-host disease, was relatively low, that B cell expression was low, and that UCB were enriched with primitive HSCs. As UCB for preterms were not significantly different from that of terms, the UCB from preterm babies might be used as a source of HSCs. Moreover, the cell number for adequate engraftment might be inferred from calculating mononuclear cells in UCB as the mononuclear cell count had a good correlation with CFUs.