Telomerase upregulation in different cytokine combination supported cells in liquid culture. (A) Representative TRAP of telomerase regulation in CD34⁺ cells in response to single cytokines and cytokine combinations on days 3, 5, and 7 of ex vivo expansion culture. Telomerase activity was maximally increased in cultures supported with K36EG or KL + IL-1 as compared with considerably lower activity using single cytokines. (B) Summary of telomerase activity (given in percentage of the neuroblastoma cell line control) of CD34⁺ cells showing upregulation of telomerase in response to various single and multiple cytokine combinations. Stimulation of CD34⁺ cells with single cytokines such as KL, IL-1, and IL-3 or combinations of KL + Flk-L or KL + IL-6 + sIL-6R displayed no or little telomerase upregulation. Using cytokine combinations of KL + IL-1 or multiple cytokines (K36GE, F36GE, KF36GE) that initiate cell proliferation and mitotic stimulation resulted in a considerable telomerase increase in Delta culture. Highest telomerase upregulation was seen using KF36EG. TGF-β1 in combination with stimulatory cytokines (K36EG) downregulated telomerase activity compared with the five factor cytokine combinations (K36EG) alone. (C) Cell expansion of CD34⁺ cells in ex vivo expansion culture using single and multiple cytokine combinations. Cell expansion was highest in multiple cytokine combinations. TGF-β1 in combination with stimulatory cytokines (K36EG) downregulated cell expansion. Combinations of KL + IL-1, KL + IL-6, KL + IL-6 + IL-6R, KL + Flk-L, or single cytokines induced only a minor cell expansion.