Abstract
Background. Umbilical cord blood (UCB), a source of hematopoeitic stem cells (HSC), is marked by delayed engraftment attributed to the limited cellular content of a single UCB unit. Cytokine-based ex vivo expansion of UCB is a way of increasing the number of cells available for allogeneic transplants, however, this strategy has not demonstrated improved engraftment in human clinical trials. Further studies have incorporated human mesenchymal stem cells (huMSC) which may provide signals that control the proliferation, survival, and differentiation of HSC. In attempt to reduce the occurrence and severity of GVHD following allogeneic transplants, strategies such as utilizing T-cell depleted grafts have been pursued, however, clinical trials using these grafts have shown decreased rates of donor engraftment, suggesting the requirement of accessory cells as well as HSC to achieve engraftment.
Methods. UCB mononuclear cells (MNC) were cultured using cytokines (IL-3, IL-6, G-CSF, SCF, Flt-3L, EPO) with or without a feeder-layer of huMSC for 12 days. On day 12, viability, 4-color flow cytometry, and human engraftment potential were measured. Human engraftment potential was determined by injecting cells (without CD34+ selection) from each culture condition and non-cultured UCB MNC, via tail vein, into sublethal irradiated NOD/SCID mice. Mice were injected with unexpanded UCB MNC (n=23), UCB expanded in huMSC+cytokines (n=21) and UCB expanded in cytokines alone (n=10). 7–9 weeks following injection of human cells, bone marrow was harvested and analyzed for human content. Positive human engraftment was determined by a human %CD45+ of ≥ 0.4%.
Results. An 8.77 fold expansion of UCB cultured in cytokines alone compared to a 7.14 fold expansion of UCB cultured in huMSC + cytokines was observed. Surface phenotyping of expanded UCB, and human cells emerging in the bone marrow of NOD/SCID mice following injection of cultured and non-cultured UCB are in
Table 1. Unexpanded huMSC+cytokines Cytokines % CD3 44.0 (4.40M) 2.04 (10.1M) 1.52 (4.35M) % CD56 17.0 (1.70M) 7.07 (36.1M) 3.69 (13.8M) % CD34 3.41 (.341M) 2.25 (10.9M) 3.52 (12.3M) Bone marrow of NOD/SCID mice % CD45+ 2.57 3.58 2.38 % of CD45+ co-expressing CD3 11.0 9.25 18.7 % of CD45+ co-expressing CD19 33.5 16.6 19.4 % of CD45+ co-expressing CD56 10.1 8.04 1.60
Human engraftment was seen in 13 mice which received unexpanded UCB, 10 mice which received UCB expanded in huMSC+cytokines and only 3 mice which received UCB expanded in cytokines alone. Statistical analysis, using multivariable logistic regression to determine the factors that predict engraftment, revealed that the proportions of T and NK cells present in expanded UCB correlated with engraftment. A 10% increase in the proportion of CD45+ co-expressing CD3 was associated with a 1.79 fold increase in engraftment (p=0.016), whereas each 10% increase in the proportion of CD45+ co-expressing CD56 increased the odds of engrafting by 104% (p= 0.003).
Conclusions. We observed an expansion of CD34 hematopoietic progenitors as well as a greater proportion of CD3+ cells, in expansion conditions incorporating huMSC. Additionally, we observed improved rates of engraftment in this expansion condition. Therefore, although the mechanism by which accessory cells including T and NK cells facilitate HSC engraftment is not known, we observed that the presence of accessory cells in addition to CD34 hematopoietic progenitors facilitated engraftment in NOD/SCID mice.
Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo