Stage 3: activation and plasma cell differentiation of in vitro–developed human B cells. Cells from stage 2 were transferred onto the MS40L-low monolayer and incubated in the presence of IL-2 (10 ng/mL), IL-10 (100 ng/mL), and CpG (2 μM) for 2 to 3 weeks. (A) Comparison of CD19 expression before (lighter lines) and after (darker lines) stage 3 showed an increased percentage of CD19⁺ cells but a decrease of CD19 intensity. Expression of CD86 is also shown. (B) Regardless of the promoter (MH or EEK) and transgene (GFP or b12), both plasmablasts (CD20⁻CD38⁺CD138⁻) and plasma cells (CD20⁻CD38⁺CD138⁺) were generated. The percentages shown are those of total live cells. (C) Class-switched B cells (surface IgG⁺) and memory B cells (CD19⁺CD27⁺) were also generated. The percentages shown are those of CD19⁺ cells. (D) B cells from stage 2 proliferated during stage 3 and formed clusters on the MS40L-low monolayer as shown in the phase-contrast and fluorescent images. Bar represents 25 μm. Cell clusters were visualized by a Nikon epifluorescence microscope equipped with a GFP filter set (4× objective lens). (E) A representative forward (FSC) and side (SSC) scatter graph of CD19⁺ cells before (darker traces) and after (lighter traces) stage 3 is shown. The cells after stage 3 were larger in size and more granular than those before stage 3. (F) Representative Wright stain images of plasmablasts and plasma cells as indicated by arrows. Bar represents 5 μm. For Wright stains, single cell suspensions were cytospinned onto slides, air-dried, stained, and examined on an Olympus BX-51 microscope (10× objective lens) and photographed using a Spot Digital Camera. (G) Endogenous levels of IgM, IgG, and IgA production by cells carrying no virus or the GFP transgene at the end of stage 2 and stage 3 were assayed using ELISA. ND indicates not detectable.