Advances in cellular technology in the hematology field: What have we learned so far?

Table 1 Steps taken to obtain erythroid cells from human embryonic stem cells by the formation of hemangioblasts

Step-Days	Procedure
1-3.5 prior to step 2	EB formation is stimulated by plating hESCs on ultra-low attachment plates with serum-free medium supplemented with BMP-4, VEGF165 and basic FGF; 48 h later, half of the medium renewed and accreted with SCF, thrombopoietin and FLT3 ligand; EBs are formed and induced to differentiate to hemangioblasts
2-d 0 to 10	EBs formed are dissociated with trypsin and a single cell suspension is obtained; The cells are re-suspended in appropriate medium and further put in contact with BGM with added FGF and t-PTD-HoxB4 fusion protein, where they are expanded for 10 d; The hemangioblasts are expanded enough after this period of 10 d
3- d 11 to 20	Differentiation to erythroid cells may be achieved by culturing the hemangioblasts obtained in step 2 for 5 d with the addition of BGM supplemented with Epo to the medium used in step 2; The cells are then expanded for up to seven more days on Stemline II-based medium supplemented with SCF, Epo and methylcellulose
4-d 21	Erythroid cells obtained after step 3 were suspended in IMDM with 0.5% BSA in order to be collected by centrifugation The cells are washed with IMDM and BSA and normally plated so that non-erythroid cells adhere, allowing the non-adherent to be separated by collecting the medium and centrifugation

hESCs: Human embryonic stem cells; FGF: Fibroblast growth factor; BMP-4: Bone morphogenetic protein-4; SCF: Stem cell factor; BGM: Blast-colony growth media; Epo: Erythropoietin; IMDM: Iscove modified Dulbecco medium; BSA: Bovine serum albumin.