title: Plant-produced human recombinant erythropoietic growth factors support erythroid differentiation in vitro.
abstract: Clinically available red blood cells (RBCs) for transfusions are at high demand, but in vitro generation of RBCs from hematopoietic stem cells requires significant quantities of growth factors. Here, we describe the production of four human growth factors: erythropoietin (EPO), stem cell factor (SCF), interleukin 3 (IL-3), and insulin-like growth factor-1 (IGF-1), either as non-fused proteins or as fusions with a carrier molecule (lichenase), in plants, using a Tobacco mosaic virus vector-based transient expression system. All growth factors were purified and their identity was confirmed by western blotting and peptide mapping. The potency of these plant-produced cytokines was assessed using TF1 cell (responsive to EPO, IL-3 and SCF) or MCF-7 cell (responsive to IGF-1) proliferation assays. The biological activity estimated here for the cytokines produced in plants was slightly lower or within the range cited in commercial sources and published literature. By comparing EC50 values of plant-produced cytokines with standards, we have demonstrated that all four plant-produced growth factors stimulated the expansion of umbilical cord blood-derived CD34+ cells and their differentiation toward erythropoietic precursors with the same potency as commercially available growth factors. To the best of our knowledge, this is the first report on the generation of all key bioactive cytokines required for the erythroid development in a cost-effective manner using a plant-based expression system.
authors.affiliation: Fraunhofer USA Center for Molecular Biotechnology, Newark, DE 19711, USA.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Musiychuk
authors.forename: Rajarajeswari
authors.initials: R
authors.lastname: Sivalenka
authors.forename: Jennifer
authors.initials: J
authors.lastname: Jaje
authors.forename: Hong
authors.initials: H
authors.lastname: Bi
authors.forename: Rosemary
authors.initials: R
authors.lastname: Flores
authors.forename: Brenden
authors.initials: B
authors.lastname: Shaw
authors.forename: R Mark
authors.initials: RM
authors.lastname: Jones
authors.forename: Tatiana
authors.initials: T
authors.lastname: Golovina
authors.forename: Jacob
authors.initials: J
authors.lastname: Schnipper
authors.forename: Luipa
authors.initials: L
authors.lastname: Khandker
authors.forename: Ruiqiang
authors.initials: R
authors.lastname: Sun
authors.forename: Chang
authors.initials: C
authors.lastname: Li
authors.forename: Lin
authors.initials: L
authors.lastname: Kang
authors.forename: Vanessa
authors.initials: V
authors.lastname: Voskinarian-Berse
authors.forename: Xiaokui
authors.initials: X
authors.lastname: Zhang
authors.forename: Stephen
authors.initials: S
authors.lastname: Streatfield
authors.forename: John
authors.initials: J
authors.lastname: Hambor
authors.forename: Stewart
authors.initials: S
authors.lastname: Abbot
authors.forename: Vidadi
authors.initials: V
authors.lastname: Yusibov
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular / Q000379:methods; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D005312:Fetal Blood / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D007377:Interleukin-3 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D019089:Stem Cell Factor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics; D019076:Transgenes
chemical_list: C554270:EPO protein, human; D007377:Interleukin-3; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D004921:Erythropoietin; D007334:Insulin-Like Growth Factor I