A common bipotent progenitor generates the erythroid and megakaryocyte lineages in embryonic stem cell–derived primitive hematopoiesis by Olena Klimchenko, Marcella Mori, Antonio DiStefano, Thierry Langlois, Frédéric Larbret, Yann Lecluse, Olivier Feraud, William Vainchenker, Françoise Norol, and Najet Debili Blood Volume 114(8):1506-1517 August 20, 2009 ©2009 by American Society of Hematology

Coculture of human H1 hES cells with the OP9 stromal cell line results in simultaneous generation of mature erythroid and megakaryocytic cells. Coculture of human H1 hES cells with the OP9 stromal cell line results in simultaneous generation of mature erythroid and megakaryocytic cells. (A) Kinetics of erythroid maturation studied by flow cytometry. Embryonic (top panel) and adult (bottom panel) erythroid cells, obtained from the differentiation of mobilized CD34+ cells, cultured in the presence of SCF, EPO, and IL-3, were analyzed for the expression of GPA, CD36, and CD71 erythroid differentiation markers. The cells were also incubated with isotype-matched irrelevant antibodies as negative controls. 7-AAD–positive cells were excluded from the analyses by appropriate gating. Figure shows representative diagrams of 3 repeated determinations derived from H1 ES cell line. Values in each quadrant are the mean ± SD of 3 independent experiments. (B) Globin chains expression in hES-derived erythrocytic cells. On the left, histograms show mRNA expression of globin chains obtained using the quantitative PCR assay performed in triplicate. The error bars represent SD. The level of globin chain expression is illustrated. On the right, results are presented with respect to their gene locus. Bars represent expression levels of indicated transcripts relative to the overall expression of the α- or the β-gene locus. Olena Klimchenko et al. Blood 2009;114:1506-1517 ©2009 by American Society of Hematology

MK maturation derived from H9 hES cell. MK maturation derived from H9 hES cell. (A) CD41 and CD42 flow cytometry analysis. Expression of CD41 and CD42 MK differentiation markers, shown, respectively on the y-axis and x-axis of the dot plot, were analyzed in embryonic (left) and adult (right) megakaryocytic cells. As previously described in Figure 1A, the cells were incubated with isotype-matched irrelevant antibodies as negative controls, and dead cells were excluded from the analyses. Representative histograms and values in each quadrant are the mean ± SD of 3 independent experiments. (B) May-Grünwald-Giemsa-stained cytospins of CD41+CD42+ subsets isolated from a hES/OP9 coculture from day 14 to day 16. Various levels of maturation were observed in immature MK (first on the left) compared with mature MK (last on the right). (C) Light microscopy image of proplatelet formation in serum-free cultures. (D) Immunofluorescent staining of an MK with anti-VWF polyclonal antibody (green) and phalloidin (red) showed a granular pattern of labeling for VWF. MKs were left to adhere on polylysine-coated slides for immunolabeling and detection of cytoskeletal components. The images were acquired using a Zeiss laser scanning microscope (LSM 510; Carl Zeiss) or an inverted Leica DM IFBE microscope (Leica Microsystems) with a 63×/1.0 NA oil objective. (E) Ploidy distribution was measured by flow cytometry after staining hES-derived MKs with propidium iodide from day 14 to 16, CD34+ cord blood–derived MKs at day 10, and CD34+ leukapheresis-derived MKs at day 10. Olena Klimchenko et al. Blood 2009;114:1506-1517 ©2009 by American Society of Hematology

Kinetics of GPA, CD41, CD34, CD43, and CD42 expression during hematopoietic differentiation of H1 hES cells. Kinetics of GPA, CD41, CD34, CD43, and CD42 expression during hematopoietic differentiation of H1 hES cells. Flow cytometric analysis of the expression of GPA (y-axis) and CD41, CD42, CD43, and CD34 (x-axis) was performed in hES cell differentiation culture. Plots represent the evolution of the respective proportions of different cell populations within the GPA-positive cells. The gates identifying cells expressing GPA alone or coexpressing CD41 and GPA were set to include only 7-AAD–negative cells. Gating was settled according the negative controls (not shown). Values in each quadrant are the mean ± SD of 3 independent experiments. Olena Klimchenko et al. Blood 2009;114:1506-1517 ©2009 by American Society of Hematology

Differential expression of CD41 and GPA permits subfractionation of a compartment of primitive embryonic erythro-megakaryocytic progenitors. Differential expression of CD41 and GPA permits subfractionation of a compartment of primitive embryonic erythro-megakaryocytic progenitors. (A) Sorting strategy used to fractionate the erythro-MK progenitors. H1 hES cells were analyzed by flow cytometry after 12 days of differentiation. Cells within the CD41+ cell population were examined for GPA coexpression and subdivided into CD41+GPA+ (R1), CD41−GPA+ (R2), CD41+GPA− (R3), and CD41−GPA− (R4) cell fractions. (B) Colony-forming potential of the different GPA/CD41 cell populations derived from H1 hES cells. Cells sorted at day 9, 12, and 14 were seeded into fibrin clot culture and tested for their colony-forming potential. Cultures were allowed to grow for 7 to 10 days, fixed, and stained with an anti-CD42 antibody. Colonies were scored under an inverted microscope for Ery-P (erythroid colony), MK (megakaryocytic colony), mixed E-MK (bipotent erythroid/MK), CFU-M (macrophage colony), and CFU-GM (granulo-macrophagic colony). Data represent the mean value ± SD from 3 experiments. Although mixed E-MK colonies were present in all tested subpopulations, multivariate analysis of variance showed that the highest frequency was found within the GPA+CD41+ fraction (P < .001). (C) Analysis of the cell composition of indivdual clones derived from H9 GPA+CD41+ cells. GPA+CD41+ cells were sorted after 12 days from hES /OP9 cocultures and seeded on MS5 feeder layer at 1 cell per well, in medium containing hES-tested FBS and a combination of SCF, EPO, TPO, and IL-3. The results presented are those obtained from 75 clones analyzed at day 7 and day 12 of culture after labeling with directly conjugated anti-GPA and anti-CD41 monoclonal antibodies. (D) Semisolid assays. (Top panel) Immunostaining of fibrin clot colonies grown in fibrin clots in the presence of a cocktail of cytokines. The primitive erythroid colony (Ery-P), mixed erythro-megakaryocytic colony (MEP), and pure megakaryocytic colony (CFU-MK) were stained with an anti-CD41 Ab (pink). (Bottom panel) Methylcellulose cultures showing typical primitive erythroid colonies (Ery-P) and mixed E-MK colonies containing erythroblasts and megakaryocytes (MEP). Colonies with typical morphologic features of BFU-E obtained from the double-negative population are clearly different from Ery-P–derived colonies. Colonies were examined under a Zeiss laser scanning microscope (LSM 510; Carl Zeiss) equipped with a 63×/1.4 numerical aperture (NA) oil objective (original magnifications 20× and 10×; MEP in fibrin clot and BFU-E). Olena Klimchenko et al. Blood 2009;114:1506-1517 ©2009 by American Society of Hematology

Embryonic MEPs coexpress erythroid and megakaryocytic genes. Embryonic MEPs coexpress erythroid and megakaryocytic genes. (A) Relative expression levels of 12 genes as assessed by quantitative RT-PCR. Genes were classified according to their restricted expression in the erythroid (EPOR and KLF1) or megakaryocytic (AML1, FLI1, GABPA, and MPL) lineages (top panel). The second group was composed of genes common to the 2 types of differentiation (GATA1, NFE2, TAL1, GATA2, MYB, and LMO2). Relative expression levels (fold change) of GPA+CD41+ (MEP) and GPA−CD41+ (MK) cells were reported to the expression levels measured in the GPA+CD41− (Ery) population. Values represent the mean ± SEM of at least 3 independent experiments. Details of the protocol for this analysis are specified in “Single-cell quantitative real-time PCR.” *Significant difference (P < .05). (B) Relative expression levels of MYB, GATA2, LMO2, and KIT transcripts in embryonic MEP versus cord blood and adult progenitor cells (CD34+CD41+CD42−). Values were reported to expression levels found in the adult CD34+CD41+CD42− fraction and represent the mean ± SEM of at least 3 independent experiments. Olena Klimchenko et al. Blood 2009;114:1506-1517 ©2009 by American Society of Hematology

Expression of EPOR and FLI1 discriminates the progressive commitment of progenitor cells in the GPA+CD41+ cell fraction. Expression of EPOR and FLI1 discriminates the progressive commitment of progenitor cells in the GPA+CD41+ cell fraction. (A) Scatter plot indicating transcript levels for KLF1, EPOR, FLI1, and MPL in Ery, MEP, and MK fractions. Each dot represents the absolute number of transcripts present in each cell. Horizontal bars represent the average levels for each gene in the various populations calculated considering the log normal distribution of the data (Shapiro-Wilk normality test run to confirm that the transcript distribution is log-normal at 95% significance level; P = .05). The absolute number of transcripts for each gene was retrieved by comparing threshold cycle values of the cDNAs to those obtained from a dilution series of a well-defined quantity of a purified PCR product. (B) Unsupervised cluster analysis (K-means) of expression of EPOR, MPL, KLF1, and FLI1 in Ery, MEP, and MK fractions at a single-cell level. The gene transcript values of the 4 genes in the overall fractions were integrated together to assess for independent clustering in specific patter of expression. Three clusters were identified: cluster Ery, cluster MEP, and cluster MK (top panel). Names were given according to the main cell type represented in each cluster (cluster Ery with 52% of cells GPA+CD41−, cluster MK with 64% of cells GPA−CD41+). The cluster MEP was equally represented by the GPA+CD41−, GPA+CD41+, and GPA−CD41+ cell subsets. Profiles of gene expression were derived to describe each individual cluster (bottom panel). (C) Dot plot distribution between EPOR and FLI1 at a single-cell level. Spots from Ery, MEP, and MK clusters were colored differently. Each spot represents a single cell. Here, 3 clouds of points are observed, where cells in the MEP cluster progressively commit to the Ery or MK cluster by increasing the number of copies of EPOR or FLI1 transcripts, respectively. Olena Klimchenko et al. Blood 2009;114:1506-1517 ©2009 by American Society of Hematology

Erythropoietin is absolutely required for human primitive embryonic erythropoiesis. Erythropoietin is absolutely required for human primitive embryonic erythropoiesis. (A) Methylcellulose colony assay in various growth factor combinations. GPA+CD41+ cells were sorted at day 12 of hES/OP9 coculture and tested for colony-forming activity in the presence of different combinations of cytokines, as indicated. Cytokines were used at the following concentrations: EPO, 2 U/mL; TPO, 50 ng/mL; SCF, 50 ng/mL; and IL-3, 100 U/mL. Colonies were scored at day 9 of culture. The mean ± SD of 3 experiments is presented. (B) Morphology of Ery-P colonies grown with EPO alone (left) or with EPO + IL-3 (right). Note the difference in colony sizes. Olena Klimchenko et al. Blood 2009;114:1506-1517 ©2009 by American Society of Hematology