Maintenance of Earlier Hematopoietic Stem Cell (HSC) Phenotype and Improved NOD/SCID Engraftment for UCB Expanded Short-Term in Cytokines over a Human Mesenchymal Stem Cell (huMSC) Platform.
Authors: Kozik M. , Banks J. , Fanning L. , Finney M. , Huang Y. , Masri O. , Haynesworth S. , Koc O. N. , Szekely E. , Daum-Woods K. , Laughlin M. J. 
Published: October 2019

Abstract
Cytokine-based expansion of umbilical cord blood (UCB) in vitro prior to infusion has been pursued in an attempt to overcome the limited cellular content of a single UCB unit. Thus far, these attempts have not shown improvement in kinetics of donor-derived hematopoietic recovery. Our studies have incorporated UCB expanded over a feeder-layer of human mesenchymal stem cells (huMSC), known to inhibit the differentiation of hematopoietic stem cells (HSC) observed in expansion with cytokines alone. Expansion conditions included: UCB expanded over a huMSC monolayer with the addition of cytokines (IL-3, IL-6, G-CSF, SCF, FLT-3L, EPO) and UCB expanded in the same cytokines alone. Day 12 culture readouts included: viable cell counts, 4-color flow analysis, and rates of human engraftment in NOD/SCID mice. In the current study the fold expansion was 6.4 fold in the huMSC + cytokines condition and 7 fold in the cytokines alone condition. Flow cytometry surface marker analysis proportions (absolute numbers) were notable for higher proportions and numbers of early HSC expressing CD133 in cultures incorporating huMSC stromal layer:
Unexpanded MSC+ cytokines Cytokines CD34 0.68 (.068M) 0.74 (3.63M) 1.94 (5.39M) CD133 5.69 (.569M) 2.56 (12.54M) 0.74 (2.06M) CD3 49.6 (4.96M) 2.2 (10.78M) 0.42 (1.17M) CD56 17.4 (1.74M) 2.71 (13.28M) 1.06 (2.95M) CD69 0.80 (7.28M) 7.28 (35.67M) 24.4 (67.8M)
UCB graft T and NK populations were maintained in huMSC culture conditions and the observed difference in CD69 expression supports the hypothesis that huMSC may have an inhibitory effect on T cell activation during UCB ex vivo expansion. To assess the human engraftment potential of the cultures, cells from each culture condition were injected by tail vein into NOD/SCID mice (no CD34 selection was performed). Mice receiving unexpanded UCB received 10M mononuclear cells each. Mice receiving culture expanded cells received cell doses in proportion to the fold expansion over the number of cells at the initiation of the cultures. Engraftment was assessed by the percentage of human CD45+ (≥0.4%) cells found within the bone marrow of mice at seven weeks post infusion. Mice were injected as follows: 7 mice with unexpanded UCB (2 of which died within a month of transplant), 7 mice with UCB expanded in huMSC + cytokines, and 3 mice with UCB expanded in cytokines alone. Flow analysis of mouse bone marrow cells revealed average CD45+ percentages of 1.79% for mice injected with unexpanded UCB, 2.66% for mice injected with cytokine alone cells, and 5.94% for mice injected with huMSC + cytokine cells. Human cell subset analysis was performed for CD3, CD19, and CD56 content. The percentages of gated CD45+ co-expressing CD3+ were 10.3% in the unexpanded UCB, 16.6% in the cytokine alone condition and 10.4% in the huMSC + cytokine condition. Cells co-expressing CD19+ were 7.86% in the unexpanded UCB, 8.31% in the huMSC + cytokine condition and dropped to 1.43% in the cytokine alone condition. Gated CD45+ cells co-expressing CD56+ were 16.4% in the unexpanded UCB, 8.8% in the huMSC + cytokines condition, and dropped to 2.6% in the cytokines alone condition. In conclusion, UCB expanded short-term in cytokines demonstrates maintenance of earlier HSC phenotype and improved human engraftment in NOD/SCID in cultures incorporating a huMSC monolayer platform.