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. 2020 Dec 10;16(1):182–197. doi: 10.1016/j.stemcr.2020.11.008

Figure 3.

Figure 3

Scale-Up of Differentiation of X13 hiPSC in 125 mL Spinner Flasks

(A) Schematic of suspension culture continuous agitation differentiation process from hiPSC to erythroblast stage in 125 mL spinner flasks. Corresponding images of cell aggregates/cells during differentiation are shown below.

(B and C) (B) Concentration of viable cells during pluripotent expansion stage. (C) Flow cytometry evaluation of pluripotency markers (Oct-4, Tra1-60, SSEA-4), mesoderm/primitive streak marker (T-bra), and hematopoietic-fated mesoderm marker (KDR+) (representative experiment shown).

(D) RT-PCR quantified fold expression of early markers (GAPDH normalized) involved in hematopoietic induction at day 3 of differentiation.

(E) Time course of marker expression for hemogenic endothelial cells (CD34+ CD144+), hematopoietic progenitor markers (CD34+ CD43+; CD34+ CD45+), and pan-hematopoietic markers (CD34− CD43+; CD34− CD45+) during the course of differentiation.

(F) RT-PCR quantified fold expression of markers upregulated during erythropoiesis at day 25 of differentiation relative to undifferentiated cells.

(G) Time course of marker expression (determined by FACS) during the differentiation process from day 14 to 28. Data in (B, D, E, F, G) represent the mean ± SD with at least three independent replicates. Representative experiment shown.

(H) Cumulative fold expansion of cells during pluripotent expansion (day −8 to day 0), mesoderm induction (day 0 to day 3), hematopoietic induction (day 5 to day 14), and erythroid expansion (day 15 to day 28) stages, in three independent spinner flasks (p > 0.05 indicates three independent spinner flasks are equivalent). Corresponding hemoglobinized cell pellets at day 28 of differentiation are shown.

(I) Concentration of viable cells and corresponding viability in three independent spinner flask experiments during differentiation are shown. Black arrows indicate complete medium change. Statistical tests were performed on the pooled data from at least three independent replicates.