HOXB4 Promotes Hemogenic Endothelium Formation without Perturbing Endothelial Cell Development

Abstract

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro, is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1^(-/-) ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of hemogenic endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression.

Keywords: EHT; HOXB4; RUNX1; hemangioblast; hematopoietic specification; hematopoietic stem cells; hemogenic endothelium; pluripotent stem cells.

Figure 1

HOXB4 Does Not Promote Early Mesoderm Specification, In Vitro (A) Scheme depicting the gammaretroviral, FMEV-based expression vectors (Lesinski et al., 2012, Schiedlmeier et al., 2007). The vectors co-express a fluorescent protein (eGFP, mPlum, or tdTomato) together with HOXB4 or a 4-hydroxytamoxifen (Tam) inducible form, HOXB4^ERT2. Co-translational separation of the proteins is mediated by the TAV-2A esterase. LTR, long terminal repeat; wPRE, woodchuck hepatitis virus posttranscriptional regulatory element. (B) Vector-transduced GFP-Bry ESCs (mPlum +/− HOXB4) were differentiated as embryoid bodies (EBs). At the indicated days, eGFP fluorescence as well as FLK-1 expression were determined by flow cytometry. The percentages of eGFP⁺FLK-1⁺ cells are shown. CCE ESCs were used as eGFP-negative controls (top row). (C) After 6 days of differentiation, GFP-Bry EBs were dissociated and 10⁵ cells each co-cultured on OP9 stoma cells for further 8 days. Contour plots of flow cytometry analysis are shown, the percentages of CD41⁺ and CD45⁺ cells are indicated. For (B) and (C), representative results of n = 3 independent experiments are shown.

Figure 2

Formation of HE Colonies Is Promoted by HOXB4 (A) During co-culture on OP9 cells, circular sheet colonies were formed by the dissociated CCE-ESC-derived EBs (eGFP-HOXB4 transduced), which were commonly associated with hematopoietic suspension cell clusters. Left panel: phase contrast; right panel: eGFP-fluorescence. Scale bars, 100 μm. (B) The observed endothelial colonies expressed VE-cadherin, CD31, and were capable of acetylated low-density lipoprotein (LDL) (DilAcLDL) uptake. Scale bars, 100 μm. (C) The number of endothelial CD31⁺ and DilAcLDL⁺ colonies strongly increased when HOXB4 was ectopically expressed. Average colony numbers per 10⁵ seeded cells are represented as columns, error bars represent SD of n = 3 independent experiments. (D) iRunx-ESCs with and without a 4-hydroxytamoxifen (Tam) inducible form of HOXB4 (vector FMEV-tdTomato-2A-HOXB4^ERT) were differentiated as EBs for 6 days, dissociated, and co-cultured on OP9 stroma cells for further 4 days without Runx1 induction (no addition of doxycycline); n = 9 and 4 independent experiments for controls, n = 7 for HOXB4. Without HOXB4 induction, the number of HE colonies per 10⁵ seeded EB cells was comparable with unmanipulated controls. When HOXB4 was induced throughout differentiation, the number of HE colonies increased approximately 30-fold (p < 10⁻⁴). The p values were calculated using the two-sided, unpaired Student's t test with a significance level defined as 0.05. (E) Flow cytometric analysis showing the proportion of CD41⁺ and CD45⁺ cells in OP9 co-cultures after 5 and 12 days. Dissociated iRunx EBd6 were co-cultured on OP9 cells with or without addition of doxycycline (0.1 μg/mL) to induce Runx1 expression and with or without addition of 100 nM Tam for induction of HOXB4^ERT (FMEV-tdTomato-2A-HOXB4^ERT). Induction of Runx1 and HOXB4^ERT started from day 3 of EB development on until day 5 of OP9 co-culture. Cells were harvested after 5 or 12 days of OP9 co-culture, and the proportion of CD41- and CD45-expressing cells determined by flow cytometry. OP9 cells were removed with an anti-CD140b antibody.

Figure 3

HOXB4 Does Not Alter Blast Colony-Forming Cell (Hemangioblast) Frequencies (A) Depicted is an overview of FLK-1⁺ hemangioblast frequency determination, subsequent HE quantitation, and evaluation of tube formation propensities. iRunx cells were differentiated as EBs for 3.5 days, FLK-1⁺ cells with and without ectopically expressed HOXB4 were sorted, and 50,000 cells each subjected to blast colony-forming assays to retrospectively quantify the number of hemangioblasts 4 days later. All colonies were harvested, dissociated, and 50,000 cells each placed onto OP9 stroma cells to determine the number of HE colony-forming progenitors 3 days later. A total of 40,000 cells each were placed into a Matrigel-based tube formation assays. All assays were performed at least in triplicate, without Runx1 induction. (B) Ectopic HOXB4 expression did not significantly alter the total number of colonies. For statistical analysis, p values were calculated based on the two-sided, unpaired Student's t test, n = 6–12; the significance level was defined as p < 0.05. The individual colony numbers are shown as symbols, the arithmetic means depicted as lines. (C) HOXB4 altered the ability of blast colony cells to form tubes. Instead of the small, thin tubular network observed in the controls (Ctrl and HOXB4^ERT without Tam), flat, adherent structures were formed with differing morphologies. The mid-panels show magnifications of the areas indicated in the left pictures. Scale bars, 50 μm. (D and E) Frequencies of BL-CFCs (arithmetic means, as described in B) (D) and of HE colonies (arithmetic means of n = 3 independent replicates) without HOXB4 induction (HOXB4^ERT no Tam) or with constitutively expressed (HOXB4 const) or induced HOXB4 (HOXB4^ERT + Tam) (E).

Figure 4

The Progeny of Hemangioblasts Is the Prime Target of HOXB4 Activity (A) Ectopic HOXB4 activity was induced by addition of Tam at the indicated stages of iRunx-ESC differentiation. BL-CFC assays were performed with 10,000 FLK-1⁺ iRunx EBd3.5 cells each, +/− Tam. (B) The total number of colonies was counted 4 days later, the average numbers of n = 3 independent experiments are indicated. +,+,+, Tam (HOXB4) was continuously present; −,−,−, without Tam (no HOXB4 induction); −/−/+, HOXB4 was first induced after dissociation of colonies; −/+/+, HOXB4 was first induced in EBd3.5 FLK-1-sorted cells; −/+/−, HOXB4 was only transiently induced during blast colony formation by FLK-1⁺ hemangioblasts. (C) Individual colony numbers and arithmetic means (bars): −/−, no Tam; −/+, induction of HOXB4 in FLK-1-sorted cells; +/+, continuously induced HOXB4. For statistical analysis, an unpaired, two-sided t test with Welch's correction (n = 6) was performed. The p values of individual comparisons are indicated with a significance level defined as p < 0.05. To evaluate HE formation, 10,000 cells of pooled, dissociated colonies were placed onto OP9 stroma cells +/− Tam. The numbers of HE colonies grown on OP9 cells after 3 days are shown.

Figure 5

HOXB4 Does Not Re-specify Endothelial Cells to a Hematopoietic Fate (A) iRunx ESCs expressing tamoxifen-inducible HOXB4 (tdTomato-2A-HOXB4^ERT) were differentiated as EBs for 4 days, dissociated, and single FLK-1⁺PDGFRα⁻ cells deposited and co-cultured with OP9 cells in 96-well plates. After a further 4 days, CD31⁺ tdTomato/HOXB4⁺ colonies were counted and qualified either as "endothelial colony" or "HE colony." (B) Typical colony morphologies. Scale bars, 100 μm. (C) Quantification of endothelial and HE colonies formed by single deposited cells +/− HOXB4 induction. –Tam, without HOXB4^ERT induction; +Tam, permanent induction of HOXB4^ERT. +Tam post sort, induction after single-cell deposition. The p values as calculated by Student's t test, paired and two-sided, with a significance level defined as 0.05, are indicated above. Forms and colors indicate endothelial and HE colonies grown on the same 96-well plate.

Figure 6

Gene Expression Profiling of FLK-1⁺PDGFRα⁻ Cells (A) Heatmap showing the top 32 genes (fold-change ranked) either up- or downregulated in FLK-1⁺PDGFRα⁻ cell populations from iRunx day 4 EBs without (ctrl) or with ectopically expressed HOXB4. Genes were considered differentially expressed if they met two criteria: q ≤ 0.2 and fold-change ≥ 2.0 (499 genes) (for full list see Table S1). (B) Heatmap for the expression of heptad transcription factors. (C) GSEA using gene sets specific for mesodermal progenitors ([MPs] top panel) compared with endothelial cells (ECs), ECs compared with blood progenitors ([BPs] second panel) and endothelial cells compared with mesodermal progenitors (third panel) (Scialdone et al., 2016); E8.5 RUNX⁻ endothelial cells (RUNX1-neg. ECs) compared with RUNX1⁺ endothelial cells (RUNX1-pos. ECs, fourth panel) (Swiers et al., 2013) and E10 hemogenic endothelial cells (HECs) compared with endothelial cells (ECs, fifth panel) (Solaimani Kartalaei et al., 2015). Genes were drawn according to their rank from left (high expression in control) to right (high expression in HOXB4-expressing cells) and gene sets plotted on top with each black bar representing a gene. The enrichment score is plotted in on the vertical axis.

Figure 7

Model of HOXB4 Activity during Pluripotent Stem Cell Differentiation During mouse ESC differentiation, HOXB4 appears to promote hematopoietic commitment after the hemangioblast stage in VE-Cad⁺FLK-1⁺TIE2⁺KIT⁺CD31⁺ precursors of HE cells by upregulating the expression of key hematopoietic transcription factors, in a RUNX1-independent fashion. Importantly, our results indicate that this does not happen at the expense of endothelial cells. These results are also compatible with the notion that (at least a part of) endothelial cells develop hemangioblast-independently (Padron-Barthe et al., 2014, Ueno and Weissman, 2006).