Application of a Modified Immunomagnetic Positive Selection Method for Isolation of Human CD34+ Stem/Progenitor from Cord Blood PDF

Keywords

Hematopoietic stem cells (HSCs), CD34+ cells, Human umbilical cord blood, immunomagnetic separation, Immunophenotyping, Hematopoietic progenitors.

Abstract

Umbilical cord blood (UCB) and isolated umbilical cord blood stem cells (UCBSCs) have become an alternative source of hematopoietic progenitor cells for transplantation. The aim of this study was to test the effectiveness of some modifications of human hematopoietic stem cells isolation protocols with the intention of improving the output and viability of CD34+ cells and progenitor subpopulations progeny that can be obtained from a sample of human umbilical cord blood. By that, we contribute to current studies on the human hematopoietic stem cells (HSCs) in order to bank UCB units suitable for basic research of very long- term hematopoietic as well as for transplantation. Cord blood samples were transformed to buffy coat prior to the isolation of HSCs which was performed by two steps involving CD34 pre-enrichment using human cord blood CD34 positive selection kit and an Immunomagnetic cell separation, targeting CD34 surface antigen. CD34+ cells were immunophenotyped by four-color fluorescence, using a large panel of monoclonal antibodies (CD34/PE, CD45/FITC, CD38/APC, CD33/Per-Cy, HLA-DR/PE, CD117/APC, CD123/Per-Cy, CD105-FITC, CD56/ PE, CD14/Per-Cy, CD19/Per-Cy and CD3/APC) recognizing different lineage or activation antigens. Our results showed that the percentage of CD34+ cells in whole human cord blood samples was 0.02% of total cells. After isolation by two-step, combining CD34 pre-enrichment and Immunomagnetic isolation, the frequency of CD34+ stem cells represented 0.65% among total MNCs and 83.53% among total isolated cells. This isolation leaded to a purity of over 95% and viability of 98.60%. In addition, we found that the percentage of CD34+ cells which are CD45+ was 83.53%, whereas CD34+CD38- cells comprised 21.70%. About 70.85% of isolated CD34+ cells were characterized by the absence of human leukocyte antigen-DR (HLA-DR). Concerning the CD117, CD33, CD123 and CD105 antigens which characterize true stem cells, we found a high expression percentage among isolated HUCB CD34+ cells (81.26%, 57.14% 47.45%, 58.52% for CD117, CD33, CD123 and CD105, respectively), while a very small number displayed markers of advanced myeloid commitment, such as CD14 (Myeloid lineage, 0.7%) and CD56 (NK-cell lineage, 4.48%), or those of lymphoid differentiation: CD3 (T-cell lineage, 5.22%), and CD19 (B-cell lineage, 1.76%). After testing 12 samples of cord blood using modified positive magnetic isolation technique, no variations in subpopulations were observed from sample to sample. We conclude that our modified technique enabled us to obtain an important proportion of primitive hematopoietic progenitors, as suggested by the absence of HLA-DR and CD38, as well as the presence of CD117, CD33, CD123, and CD105 on their surface. These cells are recognized as having long term reconstitution capacity within the human CD34+cell population.