RESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Eritropoetina/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-3/farmacologia , Nicotiana/genética , Fator de Células-Tronco/farmacologia , Agrobacterium tumefaciens/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular/métodos , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritropoetina/biossíntese , Eritropoetina/genética , Eritropoetina/isolamento & purificação , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Sangue Fetal/metabolismo , Expressão Gênica , Vetores Genéticos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/biossíntese , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/isolamento & purificação , Interleucina-3/biossíntese , Interleucina-3/genética , Interleucina-3/isolamento & purificação , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/biossíntese , Fator de Células-Tronco/genética , Fator de Células-Tronco/isolamento & purificação , Nicotiana/metabolismo , Nicotiana/virologia , Vírus do Mosaico do Tabaco/genética , TransgenesRESUMO
In this study, we have developed a robust cryohistological method that allows imaging of virtually any type of plant cell or tissue while preserving fluorescent protein signals and maintaining excellent cellular and subcellular morphology. This method involves modified fixation of plant tissues (i.e., leaves, stems, and petioles), infiltration in a sucrose gradient, freezing, and collection of cryosections directly onto a cryoadhesive tape. Using this method followed by microscopic analysis, we demonstrated a localized accumulation of green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinfiltrated with the movement-incompetent tobacco mosaic virus-based vector and systemic accumulation of GFP in plants infiltrated with the movement-competent vector. Overall, this simple cryohistological procedure reduced sample preparation time and allowed processing of tissue sections for high-resolution imaging of targeted fluorescent proteins in all plant tissues.