Improved Erythroid Differentiation of Multiple Human Pluripotent Stem Cell Lines in Microcarrier Culture by Modulation of Wnt/β-Catenin Signaling

Figure 1. Expansion of hPSC in agitated MC cultures result in reduced BPM4-based hematopoietic differentiation as compared to non-agitated static cultures. (A) Schematic showing the entire differentiation process starting from hPSC expansion on MCs (under static/agitation) to differentiation and expansion of hematopoietic precursors and erythroblasts followed by terminal maturation. All subsequent steps were performed under static conditions. (B) Images of hES3 MC aggregates following 7 days of static or 3 and 7 days of agitated culture. Flow cytometry evaluation of (C) pluripotency markers (3 or 7 days post hPSC expansion), (D) T-Bra (48 hrs post differentiation) and KDR (4 days post differentiation) expression in hES-3-MC aggregates initially expanded under static (static hPSC expansion) or agitated (3 or 7 days agitated hPSC expansion) condition during the pluripotent expansion stage. *P<0.05; P<0.001 as compared to static hPSC expansion condition. (E) Real-time RT-PCR data showing mean fold change in expression (relative to undifferentiated hES-3) of early hematopoietic specification markers (CD31, GATA2, GATA1, SCL, RUNX1) from hES-3-MC aggregates differentiated for 4 days. *P<0.05; P<0.001 as compared to static hPSC expansion condition. (F) Images of hematopoietic precursors on day 2 and day 14 post expansion in methylcellulose-based medium. Scale bar =1000 micron. (G) Total counts of hematopoietic precursors (day 14 post expansion in BGM medium following initial seeding of 1 x 10 cells) and erythroblasts (Day 14 post seeding in erythroblast expansion medium) differentiated from hES-3-MC aggregates derived from static and 3 or 7 days agitation cultures (P<0.001 as compared to static hPSC expansion condition). (H) Table summarizing flow cytometry expression (%) of CD235aCD71, total CD235a and fetal hemoglobin (HbF) expressing cells and total yield of viable cells 28 days post differentiation of hES3-MC aggregates. Corresponding P-values for comparison between static and agitation cultures as well as images of erythroblast cell pellets at termination of experiment (day 28) are shown. All data are mean ± SEM, n=3. A