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.

Table 2 Enucleation of Hemangioblasts. The technique here summarized starts by taking the hemangioblasts at the 7^th day of step 2, clarified in Table 1

Step	Procedure
1	Hemangioblasts obtained on day 7 of the procedures described in Table 1 are filtered and plated with Stemline II medium supplemented with inositol, folic acid, monothioglycerol, transferrin, insulin, ferrous nitrate, ferrous sulphate and BSA, along with penicillin-streptomycin solution
2	For 7 d, the medium described in step 1 had hydrocortisone, SCF, interleukin-3 and Epo added; Cell density was kept at 106 cells/mL
3	After the 7th day, SCF and interleukin-3 were no longer added to the medium
4	The cells were co-cultured with human mesenchymal stem cells or OP9 mouse stromal cells at different days between days 19 and 36;
	Medium was composed as described in Step 1 with the addition of Epo

SCF: Stem cell factor; Epo: Erythropoietin; BSA: Bovine serum albumin.

Table 3 Summary and comparison of protocols employed for differentiation of embryonic stem cells and induced pluripotent stem cells

Hematopoietic lineage	Culture conditions for ESCs	Culture conditions for iPSCs	Cytokines used in ESC differentiation	Cytokines used in iPSC differentiation	Ref.
Erythrocytes	Cells grown initially on mitomycin-treated Mouse Embryonic Fibroblasts; BGM and Stemline II media are used; Final step was in co-culture with OP9 cells or human mesenchymal stem cells	Cells are grown in co-culture with human fetal liver-derived feeder layer	BMP-4, VEGF165, FGF, thrombopoietin, FLT-3L, t-PTD-HoxB4 fusion protein, Epo, IL-3	IL-3, SCF, Epo, BMP-4, Insulin-like growth factor-1	[28,31,32,74]
Megakaryocytes	Cells are grown in co-culture with OP9 stromal cells	Feeder-free culture in serum-free Stemline medium or in OP9 or C3H10T1/2	IL-6, IL11 and thrombopoietin	BMP-4, VEGF, FGF, SCF, thrombopoietin, FLT-3L, IL-11	[35,75,76]
Mast cells	Cells are grown in co-culture with OP9 stromal cells	Mice iPSCs were initially co-cultured in on mitomycin-treated Mouse Embryonic Fibroblasts with LIF added. In the final step, OP9 cells were used as feeder layer	SCF, IL-6, IL-3 and FLT-3L	IL-3, IL-6 and SCF	[43,44,77]
Macrophages	Cells are grown in co-culture with OP9 stromal cells, but in an alternative method, no feeder layer was used, EBs were produced by the spinning method	Cells are grown in co-culture with OP9 stromal cells	IL-3 and M-CSF	FGF, BMP-4, VEGF, IL-3, IL-6, IL-11, SCF	[45,78]
T lymphocytes	Cells are grown in co-culture with OP9 stromal cells	Initial culture was done on irradiated SNL76/7 cells and further steps on OP9-DL1 cells	FLT-3L, IL-7 and SCF	FLT-3L, IL-5	[49-52,79]
B lymphocytes	Cells are grown in co-culture with OP9 stromal cells	Cells are grown in co-culture with OP9 stromal cells	FLT-3L, IL-7	FLT-3L, IL-3, IL-7 and SCF	[53-55,80]
Eosinophils	Cells are grown in co-culture with OP9 stromal cells	No established protocol	IL-5, IL-3, GM-CSF and Eotaxin	No established protocol	[56]
Neutrophils	Cells are grown in co-culture with OP9 stromal cells	Cells are grown in co-culture with OP9 stromal cells	BMP-4, SCF, FLT-3L, IL-6, IL-6 receptor fusion protein, thrombopoietin and G-CSF	VEGF, IL-3, SCF, thrombopoietin and G- CSF	[59,81]
Dendritic cells	Cells are grown in co-culture with OP9 stromal cells	Cell culture was feeder free, initially on mTeSR1 medium and posteriorly using X-VIVO 15 medium	GM-CSF, IL-4, TNF-α	BMP-4, VEGF, SCF, GM-CSF, IL-4	[60,61,82]
Natural killer Cells	Cells are grown in co-culture with M210-B4 cells initially and AFT024 cells in the final step	Cells are grown in co-culture with M210-B4 cells initially and AFT024 cells in the final step	IL-3, IL-7, IL-15, SCF and FTL-3L	IL-3, IL-7, IL-15, SCF and FTL-3L	[63,83]

ESCs: Embryonic stem cells; iPSCs: Induced pluripotent stem cells; FGF: Fibroblast growth factor; BMP-4: Bone morphogenetic protein-4; SCF: Stem cell factor; BGM: Blast-colony growth media; Epo: Erythropoietin; BSA: Bovine serum albumin.