Notch-induced hIL-6 production facilitates the maintenance of self-renewal of hCD34 ⁺ cord blood cells through the activation of Jak-PI3K-STAT3 pathway

Ex vivo expansion of CD34 ⁺ stem cells in contact culture between hCD34 ⁺CD38 ^-Lin ^- cord blood stem cells and human delta-likeexpressing AFT024 feeder cells revealed increased amounts of stemness-related proteins such as HoxB4, GATA2, Bmi-1, and p21 and anti-apoptotic proteins such as Bcl-2, Bcl-xL, Mcl-1, and phospho-Bad, when compared with control or noncontact culture. Production of human IL-6 (hIL-6) was markedly elevated in the culture, but was profoundly inhibited by treatment with γ-secretase inhibitor. In addition, Notch-induced activation of STAT3 was directly involved in gene expression of hIL-6 and soluble hIL-6Rα, indicating the close linkage between Notch signaling and hIL-6 production. Furthermore, depletion of soluble hIL-6 (with hIL-6specific antibodies) and inhibition of IL-6mediated signals (with a Jak1 inhibitor and wortmannin) severely affected the maintenance of self-renewal of hCD34 ⁺ cord blood cells. It was also observed that the ex vivo expanded CD34 ⁺ cord blood cells were induced to reconstitute human immune cells in nonobese diabetic mice with severe combined immunodeficiency when compared with freshly isolated CD34 ⁺ cord blood cells. Together, these results strongly demonstrate that Notch signaling in the "cell-to-cell contact" between hCD34 ⁺ cord blood and delta-likeexpressing AFT024 feeder cells facilitates maintenance of self-renewal of hCD34 ⁺ cord blood cells through direct regulation of hIL-6 production.