Cytokine combination of ‘SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3’ stimulates highest ex vivo expansion of primitive progenitors as assayed in NOD/SCID-IL2Rγ−/−mice. (A) Limiting dilution assay for the unexpanded and expanded cells. Negative engraftment was defined by less than 0.5% of human CD45+ cell engraftment in mice bone marrow (P < 0.05). (B) Ex vivo expansion of umbilical cord blood (UCB) cells. Post-thaw UCB cells (4 × 105 cells/ml) were suspended in serum-free Stemspan® media (Stemcell technologies, vancouver, BC, Canada) supplied with cytokine combinations of ‘S + T + F’,’ S + T + F + B’,’ S + T + F + BCD’ and ‘S + T + F + ABD’ respectively, inoculated on a passage 3 to 5 bone marrow-derived mesenchymal stromal cell layer in a T175 flask and cultured for 12 days. Here, the concentration of stem cell factor (S), FLT3 ligand (F), thrombopoietin (T), IGFBP1 (A), IGFBP2 (B), IGF2 (C) and ANGPTL3 (D) was 100 ng/ml, 50 ng/ml, 100 ng/ml, 15 ng/ml, 20 ng/ml, 10 ng/ml and 20 ng/ml respectively. (C) Correlation of in vivo competitive repopulating unit (CRU) functional assay and ex vivo CD34+CD38−CD90+ cell surface marker. Results expressed as mean ± standard deviation. For multiple comparisons, Bonferroni’s test was used to correct the P value for the t test. P < 0.05 → Pcorrected < 0.017 and P < 0.01 → Pcorrected < 0.003 when n = 3. *P < 0.05, **P < 0.01. ANGPTL, angiopoietin-like protein; CFU-GM, granulocyte–macrophage colony-forming units; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; TNC, total nucleated cell.