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C. Bueno et al.
the next day and one-half volume media changes were performed every other day thereafter. Hematopoietic differentiation was assessed by flow cytometry at day 9 of co-culture. Accordingly, hESC-OP9 co-cultures were treated with collagenase IV/TrypLE and cells were dissociated and filtered through a 70-μm strainer. Cell suspensions were stained with anti-mouse CD29-FITC and anti-human CD34-PE and CD45-APC antibodies. The proportion of HEP (CD34+CD31+CD45-) and total blood cells (CD45+) were analyzed within the CD29- hESC-derived cell population. Hemogenic and endothelial HEP were distinguished based on CD34 and CD43 expression.40
Culture of FACS-isolated hemato-endothelial precursors in MS5 stroma or liquid culture
Day 9 human hESC-OP9 co-cultures were dissociated as above and both CD45+ cells and HEP were analyzed. FACS-purified HEP (CD29-CD34+CD31+CD45-) were plated onto MS5 stroma or in liquid culture for 30 or 16 days, respectively, in differentiation medium with hematopoietic cytokines (50 ng/mL stem cell factor, 50 ng/mL Flt3L, 10 ng/mL interleukin-3, 20 ng/mL interleukin-7). The medium was changed every 7 days, and the emergence of CD45+ hematopoietic cells was analyzed by FACS.
Endothelial differentiation of hemato-endothelial precursors
HEP (2×104) from day 9 human hESC-OP9 co-cultures were seeded onto 0.1% gelatin-coated plates in complete EGM-2 medi- um with microvasculature supplements (Lonza) for 7 days. Cells were then fixed, permeabilized and stained with rabbit anti- human VE-cadherin (Cayman), mouse anti-human endothelial nitric oxide synthase (BD Biosciences), and mouse anti-human von Willebrand factor (DAKO) followed by Alexa 488-conjugated anti-rabbit or Cy3-conjugated anti-mouse (Jackson Immunoresearch) antibodies. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Images were obtained using an inverted fluorescence microscope. Day 7 differentiating cells were trypsinized and cell suspensions were stained with anti-human CD31-FITC and CD144-PerCP-Cy5.5 antibodies.
Results
Co-expression of A4M and MA4 does not hijack pluripotency
We showed very recently that only 45% of t(4;11)+ B- ALL patients express the reciprocal fusion A4M, whereas MA4 is consistently expressed in all t(4;11)+ B-ALL patients (Figure 1A).18-20 Here, we generated transgenic hESC lines expressing “MA4 alone”, “A4M alone” or MA4+A4M (double fusion), (Figure 1B and Online Supplementary Figure S1B). EV (control)- and MA4-hESC were established by G418 selection.9 G418-resistant EV- or MA4-expressing hESC were then transduced with A4M/dTo-expressing lentiviruses and greater than 90% transduction efficiency was achieved. Transgenic hESC lines were maintained for more than 50 passages and retained hESC-like morphology (Figure 1B, left), transgene expression (Figure 1B, right), and expression of pluripoten- cy-associated transcription factors (Figure 1C) and surface markers (Figure 1D). All hESC genotypes formed ter- atomas in NSG mice (data not shown).9,50 Thus, (co-)expres- sion of A4M and/or MA4 is compatible with hESC pluripotency.
A4M and MA4 co-operate to promote emergence of hemato-endothelial precursors and enhance blood production
Hematopoietic differentiation was assessed using two distinct and well-established differentiation systems: EB formation43,47 (Figure 2) and OP9 co-culture47,48 (Figure 3). During differentiation, a population of primitive HEP aris- es, which is responsible for further hematopoietic and endothelial commitment10,30 (Figures 2A and 3A). We investigated whether co-expression of A4M and MA4 affects hESC-derived hematopoiesis by analyzing the emergence of HEP during EB development in hESC indi- vidually expressing the single fusions or the double fusion. We observed a pronounced (~5- to 10-fold; P<0.05) increase in HEP at days 7 and 10 of development in EB expressing the double A4M and MA4 fusion over those expressing single fusions (Figure 2B, upper-left panel). We next assessed whether co-expression of A4M and MA4 influences subsequent hematopoietic commitment of HEP. The kinetics of emergence and output of both total CD45+ hematopoietic cells and CD45+CD34+ hematopoi- etic progenitors was faster (EB day 10) from double fusion-expressing hESC than from equivalent single fusion-expressing cells, achieving a 2- to 3-fold higher hematopoietic output by day 15 of EB development (Figure 2B). Furthermore, double fusion-expressing HEP massively accelerated (EB day 10) the emergence of clono- genic hematopoietic progenitors as compared to single fusion-expressing HEP (Figure 2B, bottom-right panel). According to our previous work, if the kinetics of human EB differentiation is extended, allowing for a continuum of HEP-to-blood transition, MA4-expressing human EB display enhanced HEP production coupled to impaired blood output (EB day 20) (Online Supplementary Figure S2A) and clonogenic potential (EB day 15) (Figure 2B). We con- firmed stable expression of ectopic MA4 and A4M upon EB differentiation, supporting the link between genotype and phenotype (Figure 2C).
We also investigated hematopoietic differentiation using the OP9 differentiation system (Figure 3A,B), and by plating FACS-sorted HEP in either hematopoietic liquid
Mouse transplantation and analysis of hematopoietic-endothelial engraftment
NOD/LtSz-scid IL-2Rγ−/− (NSG) mice were housed under sterile conditions. The Animal Care Committee approved all mouse pro- tocols. Briefly, cord blood-derived CD34+ hematopoietic stem and progenitor cells (3×104 cells) or cells from day 15 EB (5×105 cells) were transplanted into the bone marrow as described previously.49 Animal health was monitored throughout the entire experiment. Mice were killed 10 weeks after transplantation and cell suspen- sions were analyzed by FACS for human chimerism using anti- HLA-ABC-FITC, anti-CD31-PE, CD144-PerCP-Cy5.5, and anti- CD45-APC antibodies.
RNA- and chromatin immunoprecipitation-sequencing
Details of the RNA- and chromatin immunoprecipitation- sequencing and analysis are provided in the Online Supplementary Methods.
Statistical analysis
All data are expressed as mean ± standard error of mean. Statistical comparisons were performed using GraphPad Prism software with the nonparametric Mann-Whitney test, two-tailed P-value (with 95% confidence interval). Statistical significance was defined as a P-value <0.05.
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haematologica | 2019; 104(6)