title: Early exposure of murine embryonic stem cells to hematopoietic cytokines differentially directs definitive erythropoiesis and cardiomyogenesis in alginate hydrogel three-dimensional cultures.
abstract: HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and β-major globin), and proteins (Gata-1 and β-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development.
authors.affiliation: 1 Biological Systems Engineering Laboratory , Department of Chemical Engineering and Chemical Technology, Imperial College London, London, United Kingdom .
authors.forename: Iliana
authors.initials: I
authors.lastname: Fauzi
authors.forename: Nicki
authors.initials: N
authors.lastname: Panoskaltsis
authors.forename: Athanasios
authors.initials: A
authors.lastname: Mantalaris
mesh_terms: D000464:Alginates / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors / Q000378:metabolism; D004920:Erythropoiesis / Q000502:physiology*; D020723:Glucuronic Acid / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000502:physiology; D056945:Hep G2 Cells; D006603:Hexuronic Acids / Q000378:metabolism; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000378:metabolism; D007377:Interleukin-3 / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D051379:Mice; D024510:Muscle Development / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology; D019089:Stem Cell Factor / Q000378:metabolism; D055544:beta-Globins / Q000378:metabolism; D055543:zeta-Globins / Q000378:metabolism
chemical_list: D000464:Alginates; D017077:Culture Media, Conditioned; D016207:Cytokines; D050981:Erythroid-Specific DNA-Binding Factors; D006603:Hexuronic Acids; D007377:Interleukin-3; D051741:Kruppel-Like Transcription Factors; D019089:Stem Cell Factor; D055544:beta-Globins; C080774:erythroid Kruppel-like factor; D055543:zeta-Globins; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D020723:Glucuronic Acid