Enforced RUNX1 Expression Increased the Numbers of CD34⁺ and CD45⁺ Cells from Human Embryonic Stem Cell-Derived Embryoid Bodies.

RUNX1 is an essential gene for mouse hematopoietic development. Mouse knockout models for either RUNX1 or its binding partner core binding factor(CBFβ) are embryonic lethal and lack definitive hematopoiesis. Dominant negative inhibitors of RUNX1 protein function CBFβ-SMMHC (INV), encoded by t(16;16) and AML1-ETO, encoded by t(8;21) are seen in approximately 8% and 15% of acute myeloid leukemia cases, respectively. Recently we reported that dominant inhibition of endogenous RUNX1 function by dual promoter lentivector-expressed INV inhibited proliferation and caused G1 arrest in both human and mouse myeloid progenitors. Conversely, exogenous expression of RUNX1 in mouse primary hematopoietic stem-progenitor cells resulted in increased proliferation [DCosta 2005]. In order to determine if exogenous expression of RUNX1 would enhance HESC-derived hematopoiesis, we used conditionally-regulated RUNX1 in human embryonic stem cells (HESC) as a model system. We transduced H1 (WA01) HESC with dual promoter lentivectors expressing modified estrogen receptor (ER) fusions: RUNX1-ER (EF-RUNX1-ER/PGK-GFP) or control GFP (Ub-GFP). Microscopically GFP⁺ HESC colonies were plucked and expanded on primary mouse embryonic fibroblasts to enrich for transduced cells. During growth under “pluripotent conditions,” no changes in HESC proliferation or differentiation (as assessed by staining for pluripotent surface markers SSEA-4, Tra-160 and CD9) were observed in cells induced to express RUNX1. Transduced HESC were allowed to undergo embryoid body (EB) formation. EB, cultured with or without 4-hydroxy-tamoxifen (4HT), were harvested at selected time points, dissociated with trypsin/EDTA, counted, and immunostained for CD34 and CD45 [Zambidis 2005; DCosta 2005]. Upon EB formation, there were 2–3-fold higher percentages and numbers of transduced (GFP⁺) cells in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture induced with 4HT than in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture without 4HT. In contrast, no 4HT-dependent difference was seen in the Ub.GFP control lentivector-transduced cell cultures. At these time points, 2–4% of the GFP⁺ cells in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture containing 4HT expressed CD34 and there were similar percentages of CD45⁺ cells, whereas there were lower numbers of cells expressing these markers in the control cultures. Thus, enforced expression of RUNX1 boosted the numbers of CD45⁺ and CD34⁺ cells in EB derived from HESC.