Figure 2
Figure 2. Stage 2: in vitro development of human B cells on stromal cells. (A) HSPCs primed and transduced with either U-GFP (top panels) or MH-GFP (bottom panels) lentiviral constructs were cultured on MS5 stromal cells for the indicated periods of time. The cultures were maintained biweekly as described in “In vitro human B lymphopoiesis culture system.” GFP expression and appearance of CD19+ cells were monitored using flow cytometry. (B) Pro-B cells started to appear at 3 weeks of stage 2, as monitored by the expression of both CD19 and CD10 on the cell surface. When gates were applied to pro-B versus non–pro-B and GFP expression was analyzed in these gates, the MH promoter showed pro-B cell–specific expression, whereas the ubiquitin promoter did not discriminate between the cells. The lighter lines represent non–pro-B cells; the darker lines represent pro-B cells. (C) Similar analysis was used to compare CD11c+ dendritic cells to CD11c− cells. (D) Surface μ heavy chain was detected as early as 3 weeks (central panels) into stage 2 and increased at 4 weeks (right panels). The colored labels match the colored lines in the graphs. The percentages shown are those of total live cells. (E) Expression levels of λ and κ light chains, VpreB surrogate light chain, and μ and δ heavy chains were plotted against CD19 expression, indicating a mixture of pre-B and immature B cells at the end of stage 2. The percentages shown are those of total live cells. (F) CD19+ cells was positive for Igα at the end of stage 2 (6 weeks). When both Igα and Igβ were examined, the percentage of Igα+ cells and that of Igβ+ cells was similar. In all analyses, cells carrying EEK-b12 showed a similar phenotype to those with MH-b12 (data not shown). Unless specified, all fluorochrome-conjugated antibodies were purchased from BD Biosciences PharMingen (San Diego, CA) or BioLegend (San Diego, CA). Flow cytometry was performed on a BD FACSCalibur System (BD Biosciences, San Jose, CA), and the results were analyzed using the FlowJo software (TreeStar, Ashland, OR).

Stage 2: in vitro development of human B cells on stromal cells. (A) HSPCs primed and transduced with either U-GFP (top panels) or MH-GFP (bottom panels) lentiviral constructs were cultured on MS5 stromal cells for the indicated periods of time. The cultures were maintained biweekly as described in “In vitro human B lymphopoiesis culture system.” GFP expression and appearance of CD19+ cells were monitored using flow cytometry. (B) Pro-B cells started to appear at 3 weeks of stage 2, as monitored by the expression of both CD19 and CD10 on the cell surface. When gates were applied to pro-B versus non–pro-B and GFP expression was analyzed in these gates, the MH promoter showed pro-B cell–specific expression, whereas the ubiquitin promoter did not discriminate between the cells. The lighter lines represent non–pro-B cells; the darker lines represent pro-B cells. (C) Similar analysis was used to compare CD11c+ dendritic cells to CD11c− cells. (D) Surface μ heavy chain was detected as early as 3 weeks (central panels) into stage 2 and increased at 4 weeks (right panels). The colored labels match the colored lines in the graphs. The percentages shown are those of total live cells. (E) Expression levels of λ and κ light chains, VpreB surrogate light chain, and μ and δ heavy chains were plotted against CD19 expression, indicating a mixture of pre-B and immature B cells at the end of stage 2. The percentages shown are those of total live cells. (F) CD19+ cells was positive for Igα at the end of stage 2 (6 weeks). When both Igα and Igβ were examined, the percentage of Igα+ cells and that of Igβ+ cells was similar. In all analyses, cells carrying EEK-b12 showed a similar phenotype to those with MH-b12 (data not shown). Unless specified, all fluorochrome-conjugated antibodies were purchased from BD Biosciences PharMingen (San Diego, CA) or BioLegend (San Diego, CA). Flow cytometry was performed on a BD FACSCalibur System (BD Biosciences, San Jose, CA), and the results were analyzed using the FlowJo software (TreeStar, Ashland, OR).

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