title: Enhanced hematopoietic differentiation toward erythrocytes from murine embryonic stem cells with HepG2-conditioned medium.
abstract: Embryonic stem cell (ESC) differentiation via embryoid body (EB) formation is an established method that generates the 3 germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Previously, we have enhanced 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 mESCs without standard formation of EBs. Two predifferentiation conditions were tested: (1) mESCs cultured 3 days in standard predifferentiation medium (control) and (2) mESCs cultured 3 days in HepG2 CM (CM-mESCs). Both groups were then exposed to primary differentiation for 8 days (ELC-formation period) and 14 days of hematopoietic differentiation. Enhanced mesoderm formation was observed in the CM-mESC group with an almost 5-fold increase in ELC formation (P ≤ 0.05) and higher expression of mesoderm genes-Brachyury-T, Goosecoid, and Flk-1-compared with those of control mESCs. Hematopoietic colony formation by CM-mESCs was also enhanced by 2-fold at days 7 and 14 with earlier colony commitment compared with those of 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 CM-mESC 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 CM-mESC cultures. These data indicate that hematopoietic cells more quickly differentiate from CM-mESCs, 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, 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: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D019070:Cell Lineage; D003114:Colony-Forming Units Assay / Q000379:methods; D017077:Culture Media, Conditioned / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology* / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000378:metabolism; D004920:Erythropoiesis; D005326:Fetal Proteins / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D051379:Mice; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D055544:beta-Globins / Q000235:genetics / Q000378:metabolism; D055543:zeta-Globins / Q000235:genetics / Q000378:metabolism; D000098908:Brachyury Protein
chemical_list: D017077:Culture Media, Conditioned; D005326:Fetal Proteins; D020825:T-Box Domain Proteins; D055544:beta-Globins; D055543:zeta-Globins; D000098908:Brachyury Protein