B‐cell precursors differentiated from cord blood CD34⁺ cells are more immature than those derived from granulocyte colony‐stimulating factor‐mobilized peripheral blood CD34⁺ cells

Abstract

SUMMARYUmbilical cord blood (CB) has been widely used instead of bone marrow (BM) and peripheral blood (PB) for stem cell transplantation (SCT). However, problems of sustained immunode®ciency after CB transplantation remain to be resolved. To elucidate the mechanism of immunode®ciency, we compared the characteristics of B cells differentiated in vitro from CD34+ cells of CB with those of PB. Puri®ed CD34+ cells from CB and PB were cultured on murine stroma cell-line MS-5 with stem cell factor and granulocyte colony-stimulating factor for 6 weeks. The B-cell precursors (pre-B cells) that differentiated in this culture system, were analysed as to their immunoglobulin heavy chain (IgH) variable region gene repertoire and the expression of B-cell differentiation-related genes. CD10 + CD19 + pre-B cells were differentiated from both PB and CB. Although the usages of IgH gene segments in pre-B cells differentiated from CB and PB were similar, the N region was signi®cantly shorter in CB-derived than PB-derived cells. Productive rearrangements were signi®cantly fewer in cells of CB than PB in the third week. Among a number of B-cell differentiation-related genes, the terminal deoxynucleotidyl transferase (TdT) gene was not expressed in CB-derived cells during the culture. These results indicated that immature features of pre-B cells from CB, such as lack of TdT expression, and a short N region and few productive rearrangements in the IgH gene, might cause the delay in mature B-cell production.

Developmental Changes in the Human Heavy Chain CDR3

“…However, when the V H segment is joined in the fetal rearrangements, the exonucleolytic activity has already increased to an adult-like level, and therefore both the V H and the D5Ј coding ends present similar excision as the adults. In this study we could confirm previous reports from cultured mouse B cell precursors (37), mouse B cells (51), and human cord blood pre-B cells (36) that the ratio between productive and nonproductive rearrangements increases significantly throughout development. Moreover, if both alleles are rearranged, the ratio between productive and nonproductive rearrangements should be 2.5:1, which in our study is closest to that noted with the fetal repertoire.…”

“…Some reports do not separate the TdT activity for each junction (17,18,20). Others indicate less N nucleotide additions in the V H D junction compared with the DJ H junction (31,36), contradicting other reports of more N nucleotide additions in the V H D than in the DJ H junctions of mouse B cells precursors (21,33,37) and human preterm and full-term neonate mature B cells (11,14). In our study after separating the number of N nucleotide additions for each junction, we confirmed that in the early developmental stages the V H D junction manifests significantly more TdT activity than the DJ H junction in the nonproductive and productive repertoires.…”

Expression and methylation status of the FHIT gene in acute myeloid leukemia and myelodysplastic syndrome

“…CB was collected after full-term deliveries with informed consent approved by the Review Board of Tokai Cord Blood Bank. 24 CD34 þ and CD34 À fractions of MDS, CB and BM MNCs were separated by using Dynabeads M-450 conjugated with an anti-CD34 monoclonal antibody and DETACHaBEAD (Dynal, Oslo, Norway), according to the manufacturer's instructions. Separated samples were subjected to flow cytometry for analyzing the purity.…”

Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia

“…CD34 ϩ cells from CB were separated from MNCs by using Dynabeads M-450 conjugated with an anti-CD34 monoclonal antibody and DETACHaBEAD (Dynal, Oslo, Norway) according to the manufacturer's instructions. 48 Each separated aliquot was confirmed to contain more than 95% CD34 ϩ cells by flow cytometry (data not shown).Cytogenetic G-banding analysis was performed with standard methods. In this study, cytogenetic risk groups were determined as follows: a favorable risk group was defined by t(8; 21) or inv16; a poor risk group by t(9; 22), del5, or del7; and an intermediate risk group by normal or other karyotypes and karyotype unknown.…”