Abstract
BackgroundThe HOX genes are master regulators of embryogenesis that are also involved in hematopoiesis. HOXA9 belongs to a cluster of HOX genes that play extensively studied roles in hematopoiesis and leukemogenesis.MethodsWe established HOXA9-inducible human embryonic stem cells (HOXA9/hESCs) with normal pluripotency and potential for hematopoiesis, which could be used to analyze gene function with high accuracy. HOXA9/hESCs co-cultured with aorta–gonad–mesonephros-derived stromal cells (AGM-S3) were induced to overexpress HOXA9 with doxycycline (DOX) at various times after hematopoiesis started and then subjected to flow cytometry.ResultsInduction of HOXA9 from Day 4 (D4) or later notably promoted hematopoiesis and also increased the production of CD34+ cells and derived populations. The potential for myelogenesis was significantly elevated while the potential for erythrogenesis was significantly reduced. At D14, a significant promotion of S phase was observed in green fluorescent protein positive (GFP+) cells overexpressing HOXA9. NF-κB signaling was also up-regulated at D14 following induction of HOXA9 on D4. All of these effects could be counteracted by addition of an NF-κB inhibitor or siRNA against NFKB1 along with DOX.ConclusionsOverexpression of HOXA9 starting at D4 or later during hematopoiesis significantly promoted hematopoiesis and the production of myeloid progenitors while reduced the production of erythroid progenitors, indicating that HOXA9 plays a key role in hematopoiesis and differentiation of hematopoietic lineages.
Highlights
The HOX genes are master regulators of embryogenesis that are involved in hematopoiesis
HOXA9, a homeodomain transcription factor that plays a key role in hematopoiesis, is the gene that is most differentially expressed between CD34+ cells derived from human cord blood and human embryonic stem cells ; in particular, the gene is much more strongly expressed in the former lineage (Wang et al 2005; Domingo-Reines et al 2017)
When HOXA9 was overexpressed starting from D0 to D14, the KDR+ cells were not influenced (Fig. 2a, Fig. S2A) while the most hematopoiesisrelated populations at D8 and D14 were significantly reduced (Fig. 2b, c, Fig. S2B, S2C), indicating that overexpression of HOXA9 from the earliest stage did not hinder the induction of mesoderm, but severely blocked human hematopoiesis
Summary
The HOX genes are master regulators of embryogenesis that are involved in hematopoiesis. Hoxa9-/- HSCs cannot rebuild the blood system (Lawrence et al 2005); enforced expression of Hoxa in bone marrow HSPCs improves the production of human hematopoietic stem cells (hHSCs) and myeloid precursors (Thorsteinsdottir et al 2002), and HOXA9 overexpression in hESC improves hematopoiesis in vitro (Ramos-Mejia et al 2014). These studies indicate that HOXA9 plays key roles in hematopoiesis. Overexpression of HOXA9 in murine marrow cells leads to expansion of HSCs and committed progenitors that likely undergo transformation to acute myeloid leukemia (AML) with coexpression of Meis and Pbx, emphasizing the gene’s key role in blood physiology and pathology (Dassé et al 2012)
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