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Plant Cell‐Secreted Growth Factors for ex vivo Mass Production of Red Blood Cells
Author(s) -
Xu Jianfeng,
Wang Xiaoting,
Wright Tristen
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.lb199
Subject(s) - stem cell , haematopoiesis , microbiology and biotechnology , ex vivo , biology , glycosylation , cell growth , cell culture , chemistry , biochemistry , in vitro , genetics
Ex vivo generation of clinically available red blood cells (RBCs) from hematopoietic stem cells (HSCs) represents a promising approach for overcoming the limitations associated with the use of donor's blood, such as shortage of blood and risk of transfusion‐transmitted infections. Both expansion and differentiation of stem cells are highly reliant on growth and differentiation factors, with the activity and purity of these molecules being critical to successful research and clinical applications. The overarching goal of this project is to develop a novel plant cell‐based bioproduction platform for high‐quality (i.e. animal‐free and endotoxin‐free) erythropoietic growth factors (eGFs) that facilitate mass production of RBCs from HSCs at significantly reduced cost. In addressing the low productivity bottleneck of plant cell culture technology, this proposal aims to leverage a unique, plant‐specific, O ‐glycosylation process called “hydroxyproline (Hyp)‐ O ‐glycosylation” for de novo design and engineering of Hyp ‐ O‐ g lycosylated p eptides (HypGPs”) that presumably function as a molecular carrier in boosting secretion of conjoined eGFs from cultured plant cells. This will dramatically increase the secreted yields of plant cell‐derived eGFs and simplify the product purification process. Three key eGFs essential for HSCs expansion and differentiation: erythropoietin (EPO), stem cell factor (SCF) and interleukin 3 (IL‐3), are expressed in tobacco BY‐2 cells as fusion with a strategically designed HypGP tag. The secreted protein yields, molecular structure, and Hyp‐ O‐ glycosylation of the HypGP‐tagged eGFs are characterized. The bioactivities of individual HypGP‐tagged eGF in stimulating proliferation and differentiation of relevant hematopoietic cell lines, including TF1 erythroleukemic cell and umbilical cord blood‐derived CD34 + cell, are evaluated to establish a suitable design of HypGP tag (size and orientation) for each eGF. A scalable cell culture system fortified sorely with plant cell‐produced eGFs will be finally developed for mass production of RBCs from CD34 + HSC cells. This project provides a new cost‐effective bioproduction platform for eGFs, facilitating manufacturing of HSCs‐derived RBCs at large scale for clinical applications. Support or Funding Information Arkansas INBRE‐Research Development Grant (RDG) under Grant No. P20GM103429; National Science Foundation under Grant No. 1605564; Arkansas Biosciences InstituteSchematic of the proposed research for mass production of red blood cells (RBCs) ex vivo from hematopoietic stem cells (HSCs) with plant produced eGFs (EPO, SCF, IL‐3 and IGF‐1) All the eGFs will be engineered in plant cells (tobacco BY‐2 cell) with a Hyp‐ O ‐glycosylated peptide (HypGP) tag that function as a molecular carrier in boosting the secretion of conjoined erythropoietic growth factors (eGFs).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .