HOXA9 promotes hematopoietic commitment of human embryonic stem cells by Veronica Ramos-Mejía, Oscar Navarro-Montero, Verónica Ayllón, Clara Bueno, Tamara Romero, Pedro J. Real, and Pablo Menendez Blood Volume 124(20):3065-3075 November 13, 2014 ©2014 by American Society of Hematology

HOXA9 is expressed in the HEPs during differentiation of hESC. HOXA9 is expressed in the HEPs during differentiation of hESC. (A) Schema of hESC hematopoietic differentiation system based on EB formation. (B) Kinetics of the emergence of HEPs during the hematopoietic differentiation protocol (left). Time course expression of HOXA9 during EB hematopoietic development (right). The expression of endogenous HOXA9 correlates with the HEPs emergence throughout EB differentiation. (C) Quantitative polymerase chain reaction analysis in isolated cell populations demonstrating that HOXA9 expression is enriched in the HEP population. Gene expression is shown relative to nonhematopoietic cells. (D) A 60-fold reduction of HOXA9 expression on day 15 EB in comparison with definitive CB-CD34+ hematopoietic cells. Relative expression is shown normalized to undifferentiated hESCs. Data represent mean ± standard error of the mean for 3 independent experiments. Veronica Ramos-Mejía et al. Blood 2014;124:3065-3075 ©2014 by American Society of Hematology

Enforced expression of Hoxa9 in hESCs. Enforced expression of Hoxa9 in hESCs. (A) Schematic representation of the lentiviral vectors used. (B) Bright field and fluorescence images of colonies of empty vector (EV)-expressing and HOXA9-expressing hESCs. (C) Quantitative polymerase chain reaction showing Hoxa9 transcript overexpression in hESCs. (D) Relative Hoxa9 expression levels reached in HOXA9-hESCs as compared with the endogenous HOXA9 expression in CB-CD34+, t(4;11) leukemic blasts and the leukemic cell line MV(4;11). Relative expression is shown normalized to EV-hESCs. (E) Western blot analysis demonstrating ectopic Hoxa9 protein expression in hESCs. Veronica Ramos-Mejía et al. Blood 2014;124:3065-3075 ©2014 by American Society of Hematology

HOXA9 enhances hematopoietic differentiation of hESCs in OP9 coculture. HOXA9 enhances hematopoietic differentiation of hESCs in OP9 coculture. (A) Schema of hESC hematopoietic differentiation system based on OP9 coculture and endpoint analysis (left panel). Representative flow cytometry displaying how HEPs (CD45−CD31+CD34+) and blood cells (CD45+/CD45+CD34+) are identified within the human GFP+ population (right panel). (B) Enforced expression of Hoxa9 enhances the differentiation into primitive blood cells (CD34+CD45+) and total blood cells (CD45+). (C) CFU read out from day 15 EBs confirms a significantly increase in hematopoietic clonogenic potential of the HOXA9-hESC blood derivatives. Scoring of CFU reveals a skew toward G-CFU in Hoxa9-transduced progenitors (right pie charts). (D) Ectopic Hoxa9 remains highly expressed throughout the differentiation. Relative expression is shown normalized to undifferentiated hESCs. Data are presented as mean ± standard error of the mean for 3 independent experiments. G, granulocyte; M, monocyte; GM, granulocyte-macrophage; E, erythroid. Veronica Ramos-Mejía et al. Blood 2014;124:3065-3075 ©2014 by American Society of Hematology

Overexpression of HOXA9 during EB development leads to an increased hematopoietic differentiation. Overexpression of HOXA9 during EB development leads to an increased hematopoietic differentiation. (A) Schematic of the EB-based hematopoietic differentiation and endpoint analyses (left panel). More than 90% of the cells within both empty vector (EV)-hESC/EB and HOXA9-hESC/EBs cultures were transduced (GFP+; middle panel). Representative flow cytometry displaying how HEPs (CD34+CD31+CD45−) and blood cells (CD45+) are identified (right panel). (B) HOXA9 robustly promotes differentiation into HEPs, primitive blood cells (CD34+CD45+), and total blood cells (CD45+). (C) Hoxa9-expressing blood progeny displays threefold higher clonogenic potential (left panel). Colonies retained GFP expression after the CFU assay, and scoring of CFU reveals a skew toward G-CFU in Hoxa9-transduced progenitors (middle and right panels). (D) Quantitative PCR analysis demonstrating that exogenous levels of Hoxa9 remain high throughout EB differentiation. Data represent mean ± standard error of the mean for 6 independent experiments. Veronica Ramos-Mejía et al. Blood 2014;124:3065-3075 ©2014 by American Society of Hematology

HOXA9 promotes specification rather than survival or proliferation of HEPs. (A) Apoptosis and cell cycle analysis on empty vector (EV)- or HOXA9-hESC–derived HEPs. Similar numbers of dead cells (Annexin V+/7AAD+), apoptosis-undergoing cells (Annexin V+), an... HOXA9 promotes specification rather than survival or proliferation of HEPs. (A) Apoptosis and cell cycle analysis on empty vector (EV)- or HOXA9-hESC–derived HEPs. Similar numbers of dead cells (Annexin V+/7AAD+), apoptosis-undergoing cells (Annexin V+), and cycling cells (S, G2, and M) were found in EV-HEPs versus HOXA9-HEPs. (B) HEPs were purified at day 10 of differentiation and cocultured with OP9 for 4 days. HOXA9-HEPs differentiated faster toward CD34+CD45+, and CD45+ hematopoietic cells. Data represent mean ± standard error of the mean (SEM) for 4 independent experiments. (C) Gene expression kinetics of the mesendodermal transcription factors Brachyury and MixL1 during EV- and HOXA9-EB hematopoietic differentiation. Data represent mean ± SEM for 2 independent experiments. SSC, side scatter. Veronica Ramos-Mejía et al. Blood 2014;124:3065-3075 ©2014 by American Society of Hematology

HOXA9 silencing abrogates hematopoietic differentiation of hESC. HOXA9 silencing abrogates hematopoietic differentiation of hESC. (A) Hematopoietic differentiation of hESCs transduced with an irrelevant short hairpin RNA sequence (scramble) or 3 different short hairpin RNA specific sequences for HOXA9 (shHOXA9). (B) CFU read out from day 15 EBs confirming greater than a twofold decrease of hematopoietic progenitor potential in short hairpin (shHOXA9) cells. Scoring of CFUs revealed a skewed differentiation toward erythroid lineage in shHOXA9 progenitors. (C) Quantitative reverse-transcription polymerase chain reaction analysis showing a highly reduced HOXA9 expression throughout the differentiation in shHOXA9 EBs. Data represent mean ± SEM for 3 independent experiments. (D) Quantitative reverse-transcription polymerase chain reaction showing the expression of the pluripotency factors OCT4, SOX2, and NANOG in scramble- and shHOXA9-hEBs after 15 days of hematopoietic differentiation (upper panel). Flow cytometry confirming that shHOXA9-EBs are composed by OCT4+ cells after 15 days of hematopoietic differentiation (lower panel). mRNA, messenger RNA. Veronica Ramos-Mejía et al. Blood 2014;124:3065-3075 ©2014 by American Society of Hematology

GEP in HOXA9-expressing HEPs GEP in HOXA9-expressing HEPs. (A) Top 15 biological functions of genes differentially expressed in HOXA9-HEPs compared with empty vector-HEPs, ranked by P value. GEP in HOXA9-expressing HEPs. (A) Top 15 biological functions of genes differentially expressed in HOXA9-HEPs compared with empty vector-HEPs, ranked by P value. The top 2 biofunctions in HOXA9-HEPs are “immune cell trafficking” and “hematological system development and function.” (B) The top 20 predicted activated biofunctions (z score >3) in HOXA9-HEPs. Z score: black bars, left Y-axis; -log (P value): filled gray circle with gray line, right Y-axis. (C) Upstream transcription factor regulators predicted to be activated in HOXA9-HEPs (z score >2). Veronica Ramos-Mejía et al. Blood 2014;124:3065-3075 ©2014 by American Society of Hematology