(A) The 2D differentiation protocol. Pluripotent stem cells (PSC) grown as colonies (in orange) on Matrigel (in blue) are dissociated and plated on a matrix protein (fibronectin or collagen IV, in green). After 24 hrs growth factors are added to the medium to induce hematopoietic progenitor cell (HPC, in yellow) development. The growth factor cocktail is change after 6 days of differentiation. (B) Cell morphology during the differentiation of pluripotent stem cells. After 6 days of differentiation, cells remain adherent, but change in morphology. Only after 10 days of differentiation do some colonies of round, loosely attached cells start to appear (arrow). After 14 days of differentiation many round, loosely attached cells cover the dish on top of a layer of adherent cells. (C) Percentage of CD43⁺ cells after 6 days of differentiation on different matrix proteins. Standard errors are indicated (Hypoxia in blue: n = 20 on hFibronectin, n = 3 on hCollagen IV, n = 27 on mCollagen IV; Normoxia in red: n = 5 on hFibronectin, n = 1 on mCollagen IV). Statistically significant difference between hFibronectin and mCollagen IV is indicated with a star (*) p<0.005. The yield of CD43⁺ cells (in green) is calculated as total number of CD43⁺ cells generated per each hESC induced to differentiate under hypoxic conditions.

(A) FACS analysis of differentiated cells at day 6 (total culture), at day 10 (only floating cells analyzed) and at day 14 (only floating cells analyzed). Representative FACS plots are indicated, showing live- and CD34⁺-gated events for CD43⁺CD31⁺ cells at day 6, and live-gated events for all the other plots. (B–C) Colony assays of hESC-derived hematopoietic progenitors harvested after 6 days (total culture) and after 10 or 14 days (floating cells) of differentiation. In methylcellulose-based assay (MethoCult), day 6 hESC-derived progenitors generated salmon-pink erythroid colonies; floating hESC-derived progenitors generated CFC-GM, CFC-M, CFC-E and mixed colonies. Average CFC values per 10⁵ cells ± standard errors: Day 6 N = 9 CFC-E 84.69±32.33, CFC-M 7.68±2.54, CFC-GM 1.49±0.93, mixed 1.58±0.32; Day 10 N = 5 CFC-E 208.88±75.59, CFC-M 26±11.90, CFC-GM 2.88±1.97, mixed 3.52±0.75; Day 14 N = 8 CFC-E 117.33±52.40, CFC-M 72.63±20.28, CFC-GM 13.28±4.36, mixed 2.5±0.98. In collagen-based assay (MegaCult), hESC-derived progenitors developed large colonies of megakaryocytes capable of shedding pro-platelets (arrow). (D) Yield of hematopoietic cell subsets obtained per one differentiating hESC at day 6 (in blue), day 10 (in red) or day 14 (in green). Standard errors are indicated (CD43⁺ cells: at day 6 N = 23, at day 10 N = 11, at day 14 N = 16; CD43⁺CD34⁺ cells: at day 6 N = 18, at day 10 N = 8, at day 14 N = 16; CD45⁺ cells: N = 23, at day 10 N = 6, at day 14 N = 11).

FACS analysis and corresponding cytospins of mature cells generated in culture from hESC-derived progenitors induced with defined factors. Images were taken at 40× magnification. Cytospins of erythroid cells, dendritic cells, macrophages and neutrophils were stained with Wright. Cytospins of megakaryocytes were stained for GPIIb/IIIa (CD41a). Cytospins of mast cells were stained for tryptase. The yield hESC:mature cell indicates the number of each mature cell type generated per each hESC induced to differentiate.

(A) Percentage of positive cells for the indicated marker in the differentiating culture. Total culture at day 6 of differentiation, or floating cells at day 14 were analyzed. Standard errors are indicated (n = 10 H1, n = 3 FFiPSC 6.6, n = 2 FFiPSC 6.1, n = 3 BliPSC CK, n = 4 BliPSC NL, n = 2 KiPSC2, n = 3 KiPSC5, n = 6 SiPSC, n = 2 TiPSC 1b, n = 3 TiPSC 1ee). (B) FFiPSC 6.1-derived hematopoietic progenitors isolated at day 10 of differentiation generated CD41a⁺CD42b⁺ megakaryocytes in culture, as indicated by flow cytometry analysis, cytospins stained for GPIIb/IIIa (CD41a), and megakaryocytes colonies in collagen-based assay. The megakaryocytes were able to produce pro-platelets (arrow) visible both in culture and in collagen-based colony assay.