title: In Vitro Differentiation of Embryonic Stem Cells into Hematopoietic Lineage: Towards Erythroid Progenitor's Production.
abstract: Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.
authors.affiliation: Biological Systems Engineering Laboratory, Department of Chemical Engineering and Chemical Technology, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
authors.forename: Iliana
authors.initials: I
authors.lastname: Fauzi
authors.affiliation: Biological Systems Engineering Laboratory, Department of Chemical Engineering and Chemical Technology, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.|Department of Hematology, Imperial College London, Northwick Park & St. Mark's campus, London, HA1 3UJ, UK.
authors.forename: Nicki
authors.initials: N
authors.lastname: Panoskaltsis
authors.affiliation: Biological Systems Engineering Laboratory, Department of Chemical Engineering and Chemical Technology, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK. a.mantalaris@imperial.ac.uk.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Mantalaris
mesh_terms: D000818:Animals; D001399:Azure Stains / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D017077:Culture Media, Conditioned / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004920:Erythropoiesis; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D012313:RNA / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
chemical_list: D001399:Azure Stains; D017077:Culture Media, Conditioned; D012313:RNA
keywords: Cell culture techniques; Cell differentiation; Embryonic stem cells; Erythropoiesis; Hematopoiesis