Table 1.
Fig 1.
CD34+ generation from hPSC without cytokines and their low potential in B cell differentiation.
(A) Protocol of hPSC differentiation into B cells as previously described [14]. (B) CD34+ differentiation from H9 hESC onto OP9 stroma at Day 10. CD34/CD43 expression analyzed by flow cytometry on viable differentiated hESC is shown in the left as a dot plot from one representative experiment and the proportion of CD34+ cells (in black) and CD34+CD43+ (in red) are shown on the right panel, n = 9 experiments. (C) B cells differentiation at Day 21 from UCB CD34+ cells (Upper panel) and CD34+-derived hESC (middle panel). CD10+CD19+ B cells proportion and their IgM expression was analyzed on viable CD45+ cells, dot plot from one representative experiment is shown. Histogram of CD10+CD19+ B cells proportion is presented in the lower panel (n = 3 experiments from both UCB CD34+ cells and hESC-derived CD34+).
Fig 2.
Cytokines allow CD34+CD43+ differentiation from stem cells.
(A) Table of the cytokine conditions tested according their presence from Day 1 to Day 3 and Day 4 to Day 10 for the CD34+ differentiation (cytokines were used at 10ng/ml except for BMP4 at 5ng/ml). (B) Percentage of CD34+CD43+ cells at the end of the CD34+ differentiation according to the cytokine conditions used (n = 2 or 3 independent experiments) or without cytokines as control (Ctrl, n = 9 independent experiments). The significance of differences between the condition C and the Ctrl was determined using a Mann-Whitney Test; *p < 0.05. (C) Percentage of CD10+CD19+ B cells at Day 21 of B cell differentiation from hESC-derived CD34+ with (Condition D; n = 2 independent experiments) or without cytokine (Ctrl, n = 3 independent experiments).
Fig 3.
Cytokines at 10ng/ml permit the best B cell differentiation.
hESC (Upper panels) and hIPSC (lower panels) were used for CD34+ differentiation with our protocol of cytokines addition (the cytokine condition D, as detailed in Fig 2A) at different concentration. (A, C) Histograms show the proportion of CD34+ (left panel) and CD34+CD43+ (right panel) after CD34+ differentiation from (A) hESC with cytokines (n = 1 to 3 independent experiments) or without cytokines as control (Ctrl, n = 9 independent experiments) and from (C) hIPSC (n = 1 to 4 independent experiments) The significance of differences between the condition at 25ng/mL and the Ctrl was determined using a Mann-Whitney Test; *p < 0.05; **p < 0.01. (B, D) B cells proportion at Day 21 after B cell differentiation from the corresponding CD34+ cells is shown from (B) hESC-derived CD34+ cells with cytokines (n = 1 or 2 independent experiments) or without cytokines as control (Ctrl, n = 3 independent experiments) during CD34+ differentiation and from (D) hIPSC-derived CD34+ (n = 1 to 3 independent experiments).
Fig 4.
Gating strategy of B cell progenitors.
(A) B cell characterization during B cell differentiation of UCB CD34+ cells using CD10+CD19+ total B cells gated on CD45+ (upper panel) and using CD79a-CD10- CLP, CD79a+CD10- Pre-pro-B cells and CD79a+CD19+ both Pro-B and Pre-B cells gated on CD45+CD10+ (lower panel). (B) B cell progenitors proportion in 2 independent experiments. (C) B cell progenitors proportion at Day 7 (D7), 14 (D14) and 21 (D21) of B cell differentiation from hESC-derived CD34+ (n = 1 or 2 independent experiments).
Fig 5.
Gene expression at different time point of B cell differentiation.
Gene expression analysis by Taqman expression assay of PAX5, EBF1, VPREB1, IL-7RA, IGLL1, RAG1 and OCT4 were performed on total cells at different time point during B cell differentiation. Histograms of 2-DDCT value display gene expression analysis of cells (A) at Day 21 of B cell differentiation from UCB CD34+ cells (n = 2 to 3 independent experiments) and (B) at Day 0 (D0), 7, 14 and 21 of B cell differentiation from hESC-derived CD34+ with undifferentiated hESC and tonsil B cells (LB) as controls (n = 1 to 2 independent experiments).
Fig 6.
CD31intCD45int are important progenitors for B cells differentiation.
Characterization of CD34+ progenitors by their expression analysis of the following markers CD45, CD43 and CD31. FACS analysis was performed from (A) UCB CD34+ cells (Upper panel) with unstained cells as control (lower panel), dot plot representative of 2 experiments and from (B) hESC-derived CD34+ (Upper panel) and hIPSC-derived CD34+ (lower panel) using our CD34+ differentiation protocol with cytokines at 10ng/ml, dot plot representative of 2 experiments for hESC and 4 experiments for hIPSC. (C) Histograms show CD31intCD45int proportion in hESC (upper panel) and hIPSC (lower panel) at Day 10 of CD34+ using our CD34+ differentiation protocol with cytokines at different concentrations (n = 1 to 4 independent experiments).
Fig 7.
Day 10 of CD34+ differentiation permits the better B cell differentiation.
(A) CD34+ differentiation from hIPSC, with our protocol of cytokine addition (cytokine condition D, as detailed in Fig 2A) at 10ng/ml, was stopped at Day 8, Day 10 and Day 12 to analyze the expression of CD34+ cells for the following markers CD45, CD43 and CD31. Dot plots from one experiment for Day 8 and Day 12 and 4 independent experiments for Day 10 are shown in the left panel. Histogram of the CD31intCD45int proportion among the CD34+ at different time of the CD34+ differentiation is shown. (B) Histogram represents the B cell proportion at the end of the B cell differentiation using the corresponding hIPSC-derived CD34+ (n = 1 to 3 independent experiments).