DL1 causes the loss of mIL-6R, inhibiting IL-6 cis-signaling. (A) DL1 inhibited the rapid upregulation of mIL-6R on (i) CD34⁺CD45RA⁻ cells and (ii) CD34⁺CD45RA⁺ cells on culture of CD34⁺ cells. (B) DL1 significantly decreased mIL-6R on CD34⁺ cells throughout culture. The effect was JAK-independent. *Statistical comparison of DL1 to control condition (C) Representative flow cytometry plots at day 16 (i) without DL1 and (ii) with DL1. (D) The impact of DL1 on mIL-6R was similar on (i) CD34⁺CD45RA⁻ and (ii) CD34⁺CD45RA⁺ cells. (E) Fed-batch (FB) conditions reduced sIL-6R production as compared with non–FB conditions. (F) Protein expression of ADAM17 measured by flow cytometry on cells cultured in the absence or presence of DL1 for 10-12 days. (G) Comparison of (i) surface expression of mIL-6R (CD126) and (ii) intracellular expression of mIL-6R on CD34⁺ cells cultured in the absence or presence of DL1 for 8-12 days. (H) Schematic of proposed mechanism. DL1 activates STAT3 and causes the loss of mIL-6R. The loss of mIL-6R reduces IL-6 cis-signaling and thus reduces the production of CD14⁺ and CD15⁺. Instead, only IL-6 trans-signaling can occur, which enhances the production of other cell types, and results in a microenvironment that is more supportive for hematopoietic stem and progenitor cells. The FB conditions further reduce IL-6 trans-signaling. All error bars indicate standard deviation. n ≥ 3, P < .05.