Plant Cell Secreted Growth Factors Targeted to Ex Vivo Production of Red Blood Cells from Hematopoietic Stem Cells

Undergraduate #58
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Taylor Hill - University of Arkansas at Pine Bluff
Co-Author(s): Jianfeng Xu, Ph.D; and Xiaoting Wang

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Ex vivo production of red blood cells from hematopoietic stem cells used for blood transfusion represents one of the focuses in regenerative medicine. However, production of red blood cells demands significant quantity and high quality of growth factors, which makes manufacturing at large scale cost prohibitive. Human stem cell factor is a key growth factor that stimulates the proliferation and differentiation of hematopoietic stem cells to red blood cells. My project aims towards producing the human stem cell factor with the plant cell culture technology, which has been presented to be a propitious bioproduction platform for therapeutic proteins, because of its significant advantages in cost and safety over other eukaryotic production systems. However, low protein productivity and particularly low secreted protein yield is a common blockage towards commercialization of this production platform. In addressing this problem, the human stem cell factor was expressed in plant cell with a hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) that presumably functions as a molecular carrier in promoting efficient transport of the conjoined recombinant protein into the culture media and protecting the protein from proteolytic degradation, which ultimately boosts the secreted protein yield. In this study the human stem cell factor was expressed in tobacco BY-2 cell with a HypGP carrier comprised of 20 tandem repeats of “Ala-Pro” dipeptide motif or (AP)20 at either N-terminus or C-terminus. High expression BY-2 cell lines were screened by Dot Blotting. The produced human stem cell factor fusion proteins were characterized by Western Blotting. Finally, the kinetics of plant cell growth and human stem cell factor accumulation in culture media and inside cells were determined over a 14-day growth cycle. Up to 10.5 mg/L of secreted recombinant protein was harvested. This research may provide a promising plant cell-based platform to produce large quantity of hematopoietic stem cells that assists the stem cell research and clinical applications.

Not Submitted

Funder Acknowledgement(s): National Science Foundation

Faculty Advisor: Jianfeng Xu, jxu@astate.edu

Role: I expressed the human stem cell factor in tobacco BY-2 cell with a HypGP carrier comprised of 20 tandem repeats of ?Ala-Pro? dipeptide motif or (AP)20 at either N-terminus or C-terminus. High expression BY-2 cell lines were screened by Dot Blotting. The produced human stem cell factor fusion proteins were characterized by Western Blotting. Finally, the kinetics of plant cell growth and human stem cell factor accumulation in culture media and inside cells were determined over a 14-day growth cycle. Up to 10.5 mg/L of secreted recombinant protein was harvested.