Ex vivo expansion of hematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. In this study, we investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis on prolonged maintenance and expansion of cord blood CD34+ cells. Enriched CD34+ cells (1 x 105/mL, n=5) or mononuclear cells (1 x 106/mL, n=8) were cultured in X-VIVO-10 medium for 14, 21, 28 and 35 days without supplementary cytokine or medium changing. Our results showed that the presence of NTL (200 ng/mL) or FL-3 ligand (FL, 40 ng/mL) significantly preserved populations of early stem/progenitor cells (total CFU, BFU/CFU-E, CFU-GM, CFU-GEMM) in these cultures, compared with respective controls at various time points. In the ex vivo expansion study (n=16), the presence of stem cell factor (S, 50 ng/mL), thrombopoietin (T, 50 ng/mL), FL (F, 80 ng/mL) effectively expanded total nucleated cells (TNC) at day 8 (116 ± 20.2 fold) and day 12 (424 ± 68.8 fold), as well as all subsets of progenitor cells as demonstrated by flow cytometry and CFU assays. The presence of NTL (200 ng/mL) significantly increased TNC (148 ± 24.5 fold at day 8; 572 ± 91.9 fold at day 12; P < 0.01) and expansion of early progenitor cells (CD34+, CD34+CD38−, CFU-GEMM) and committed CFU of the myeloid (CFU-GM), erythroid (BFU/CFU-E) and the megakaryocytic lineage (CFU-MK) (P < 0.01 compared with respective TSF cultures). There was also slight but consistent increase of CD61+CD41+ cells in the presence of NTL (8.58 ± 2.14 x 105 vs. 7.30 ± 1.82 x 105 cells/mL, P < 0.001). Significantly, the increased expansion was not only contributed by the higher TNC, but also by the increase in the proportion of CD34+ cells, CD34+CD38− cells and the density of differential CFU. Six weeks after enriched CD34+ cells at day 0 or expanded cells at day 12 were infused into sub-lethally irradiated NOD/SCID mice, human CD45+ cells were detectable in the BM, spleen and PB of the mice. In the BM, there were engraftments of human hematopoietic cells of the early (CD34+), myeloid (CD33+, CD14+), B-lymphoid (CD19+) and megakaryocytic (CD61+) lineages. In animals that received day 12 expanded cells in the TSF + NTL group, there was a significant increase of human CD45+ cells in the BM (19.3% vs. 11.5%, P = 0.03, n = 15) when compared with those only exposed to TSF, and a trend of increased engraftment in their spleen (P = 0.07, n = 14). Comparison of the complete amino acid sequences of NTL and FRIL (a dicot mannose-binding lectin shown to preserve hematopoietic stem cells, PNAS, 96, 646–650, 1999) showed 10.2% identity and both peptides contain putative functional/structural sites such as those for N-myristoylation, casein kinase II phosphorylation, protein kinase C phosphorylation and N-glycosylation. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34+ cells for transplantation.
Disclosure: No relevant conflicts of interest to declare.