Rapid Mast Cell Generation from Gata2 Reporter Pluripotent Stem Cells

Abstract

Mast cells are tissue-resident immune cells. Their overgrowth/overactivation results in a range of common distressing, sometimes life-threatening disorders, including asthma, psoriasis, anaphylaxis, and mastocytosis. Currently, drug discovery is hampered by use of cancer-derived mast cell lines or primary cells. Cell lines provide low numbers of mature mast cells and are not representative of in vivo mast cells. Mast cell generation from blood/bone marrow gives poor reproducibility, requiring 8-12 weeks of culture. Here we report a method for the rapid/robust production of mast cells from pluripotent stem cells (PSCs). An advantageous Gata2Venus reporter enriches mast cells and progenitors as they differentiate from PSCs. Highly proliferative mouse mast cells and progenitors emerge after 2 weeks. This method is applicable for rapid human mast cell generation, and could enable the production of sufficient numbers of physiologically relevant human mast cells from patient induced PSCs for the study of mast cell-associated disorders and drug discovery.

Keywords: ESC; Gata2; Venus reporter; differentiation; iPSC; immune effectors; innate immune cells; mast cells; rapid protocol; stem cells.

Graphical abstract

Figure 1

G2V mESC Differentiation and VENUS Enrichment Facilitates Rapid and Robust Mast Cell Generation (A) Three-stage differentiation protocol for mast cell production. Stage 1 is hematopoietic commitment in 10-day embryoid body (EB) culture. At day 10, GATA2VENUS⁺ (V⁺) cells are sorted and plated onto OP9 cells in stage 2, where mast cell commitment is induced to yield highly proliferating mast cells that appear after 3–4 days. At day 14, V⁺ mast cells/progenitors are isolated for analysis/further clonal expansion in stage 3. (B) FACS dot plot of cells from day 10 G2V mouse EBs showing gating used for sorting V⁺ cells (1.6%) after stage 1. (C) Visible-light image of a 4-day OP9 co-culture in which round refractive mast cells are observed overlaying the adherent large OP9 cells. Scale bar, 100 μm. (D) FACS analysis of cells harvested after 4 days of OP9 co-culture. 37% ± 6.8% of cells are V⁺ and CD45⁺ (red gated area). (E) 99% ± 0.6% of V⁺CD45⁺ cells shown in (D) are CKIT⁺ hematopoietic cells. Mean ± SEM, number of independent experiments (n) = 6. (F) FACS dot plots showing lack of B-lymphoid (CD19), macrophage (MAC1), granulocyte/basophil (GR1), and erythrocyte (TER119) lineage-positive cells in V⁺CD45⁺ population after 4 days of OP9 co-culture. (G) DNA PCR data of six representative spleen colonies from CFU-S assay testing the in vivo repopulation potential of V⁺CD45⁺Lin⁻ ESC-derived cells. Sorted cells injected into irradiated mice formed a few small spleen colonies at 9 days post injection. PCR for G2V and Gata2 alleles shows that the CFU-S did not originate from the ESC-derived cells. Negative control is wild-type (wt) ESC DNA. Positive control is G2V ESC DNA. (H) FACS analysis of (top) day-10 V⁺ cells for CKIT, CD45, and FCɛR1α expression shows commitment to hematopoietic progenitors but not mature mast cells. FACS analysis of day-14 V⁻CD45⁻ cells (bottom left) and V⁺CD45⁺ cells (bottom right; 83% mast cells) isolated after stage 2. Mean ± SEM, n = 6. (I) FACS histogram of VENUS expression intensity of day-10 (black) and day-14 (red) differentiated cells. Bar graph quantification of VENUS mean fluorescence intensity (MFI) at days 10 (black) and 14 (red) showing significantly higher Venus protein levels in day-14 mast cells. Mean ± SEM, ^∗∗p < 0.01, n = 3.

Figure 2

G2V ESC-Derived V⁺ Cells Generate Mucosal and Connective Tissue-Type Mast Cells that Express Proteases and Degranulate (A) Image showing the clonal expansion capacity of a single V⁺ mast cell harvested from stage 2 after 5 days. Area within the dotted region shows part of such a colony (>1,000 cells). Scale bar, 100 μm. (B) Image of toluidine blue-stained mast cells generated after stage 3 expansion. Lighter blue indicates mucosal mast cells (mMC) and darker blue connective tissue-like mast cells (ctMC). (C) Mast cell-specific gene expression in undifferentiated ESCs (number of independent experiments [n] = 3), day-10 V⁺ hematopoietic cells (stage 1, n = 3), expanded mast cells (stage 3, n = 4) and mast cells after clonal replating (rpMC, n = 3). RT, reverse transcriptase; CPA, carboxypeptidase; mMCP, mouse mast cell protease. (D) Image of toluidine blue-stained mast cells generated after two serial clonal replatings of a single cell harvested from stage 2 co-culture. Scale bar, 100 μm. (E) Quantitative gene expression analysis of mast cell-specific genes in expanded G2V mast cells. Expression levels were normalized to 18S expression and compared with the normalized levels in control mouse ear tissue. Mean ± SEM, n = 3. (F) ELISA assay showing the relative concentration of released tryptase in the medium of c48/80-treated (5 μM) G2V mast cells compared with untreated cells. Tryptase levels were calculated based on a tryptase standard curve. Level was set as 1 in untreated sample. ^∗p < 0.05, n = 5. (G) Immunoglobulin E (IgE) activation of degranulation. Functional assay showing the percentage of β-hexosaminidase release in the supernatant of IgE activation/antigen stimulation-treated ESC-MCs and peritoneal MC (P-MC) compared with controls. β-Hexosaminidase enzymatic activity was measured in supernatants and cell pellets and percent release was calculated as described in Experimental Procedures. Unpaired t test, two-tailed p value; n = 4. Error bars represent means ± SEM.

Figure 3

Generation of Human GATA2VENUS Mast Cells (A) The differentiation approach (Figure 1A) was applied to G2V-hESC/hiPSC to generate human mast cells. V⁺ cells were sorted from three independent cell lines (day-7 G2V-hiPSC1, day-8 G2V-hiPSC2, and day-11 G2V-hESC differentiated EBs) and subjected to OP9 co-culture. After 4 days (G2V-hiPSC1) or 5 days (G2V-hiPSC2, G2V-hESC) in step 2 culture, V⁺ and V⁻ cells were analyzed for mast cell markers CKIT and FCɛRIα by FACS. Percentages of cells in gated regions are shown (n = 1 independent experiment for each cell line/time frame). (B) Bar graph showing percentages (mean ± SEM) of CKIT⁺FCɛRIα⁺ cells obtained from the VENUS⁺ and VENUS⁻ fractions of cells harvested from day-10 differentiated and 4-day co-cultured G2V-hiPSC1 cells (n = 3; p = 0.030). (C) Image of toluidine blue (left) and rapid Romanowsky (right) stained mast cells generated after 5 days of step 3 expansion culture. Scale bars, 50 μm (left) and 10 μm (right). (D) Gene expression analysis of mast cell-specific protease tryptase (TPSAB1) in undifferentiated (n = 2) and differentiated G2V-hiPSC1 (n = 3) cells. GAPDH controlled for mRNA quantity. Relative expression levels were normalized to OP9 TPSAB1 expression, which was set as 1.