• Plant cell culture is a promising production platform for high-quality growth factors. • Hyp- O -glycosylated (SP) 20 tag increases the secreted yields of SCF up to 2.5 μg/mL. • The orientation of the (SP) 20 tag substantially impacts the potency of SCF. • (SP) 20 -tagged SCF stimulates the differentiation of hematopoietic stem cell CD34+ cells. Ex vivo generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) used for blood transfusion represents one of the focuses in current regenerative medicine. However, massive production of HSCs-based RBCs requires a significant quantity of erythropoietic growth factors, making manufacturing at large scale cost prohibitive. Plant cell culture is proposed to be a promising bioproduction platform for functional human proteins in a safe and cost-efficient manner. This study exploited a proprietary technology, named HypGP engineering technology, for high-yield production of one of the key erythropoietic growth factors--stem cell factor (SCF)--in plant cell culture. Specifically, a designer hydroxyproline (Hyp)- O -glycosylated peptide (HypGP) comprised of 20 tandem repeats of the "Ser-Pro" motif, or (SP) 20, was engineered at either the N-terminus or C-terminus of SCF in tobacco BY-2 cells. The (SP) 20 tag dramatically increased the secreted yields of SCF up to 2.5 μg/mL. The (SP) 20 -tagged SCF showed bioactivity in promoting the proliferation of the TF-1 cell line, although the SCF-(SP) 20 was 8.4-fold more potent than the (SP) 20 -SCF. Both the (SP) 20 -SCF and SCF-(SP) 20 exhibited desired function in stimulating the expansion and differentiation of human umbilical cord blood CD34+ cells towards RBCs. [ABSTRACT FROM AUTHOR]