Figure 2: Runx1 together with HoxB4 or Bmi1 augments the CFU-S forming-ability of iHP cells.

(a) Similar to control bone marrow cells (BM Ctr, tdTomato⁺), SLHR-iHP cells (tdTomato⁺) form tdTomato⁺ nodules in the spleen at 12 days post-transplantation (dpt) into lethally irradiated C57Bl/6 mice (CFU-S12). Factors were singly delivered in individual pMX constructs. 1 × 10⁵ BM control cells, 5 × 10⁶ SLHR-iHP cells or 1 × 10⁶ Kit⁺ cells were transplanted per mouse. Mice analysed: BM Ctr, n=4, SLHR-iHP cells, n=20, SLHR-iHP kit⁺ cells, n=4. These are representative of three independent experiments. Scale bar, 2 mm. (b) Comparison of CFU-S12 potential of iHP cells induced by differing TF cocktails. SL-, SLB-, SLHR-, SLRH- or SLRB-iHP cells (tdTomato⁺) were transplanted into lethally irradiated SCID mice. Factors were delivered in pMXconstructs; SLHR denotes individual delivery of S, L, H and R; SLRH and SLRB denotes use of a polycistronic construct containing S, L and R in one pMX vector, together with individual delivery of either H or B in a separate construct. For SL-, SLB-, SLHR-iHP, 5 × 10⁶ cells were transplanted. For SLRH- and SLRB-iHPs, 2 × 10⁶ cells were transplanted. Mice analysed: for SL-, SLB-, SLHR-iHP, n=6, for SLRB- and SLRH-iHP, n=12 each. Scale bar: 2 mm. These are representative of three independent experiments. (c) Frequency of CFU-S12 of different iHP cells. iHP cells are named as in b. Data are shown as mean±s.d. per spleen. These data are from three independent experiments. (d) Representative FACS analysis of tdTomato⁺ cells in BM of SCID mice at 12–14 dpt. SLR factors were delivered in one polycistronic construct. For SLRB/H-iHP cells: 2 × 10⁶ cells per mouse were transplanted; for Ctr BM cells (tdTomato⁺), 2 × 10⁵ cells per mouse were transplanted. Percentage of tdTomato⁺ cells are shown as mean±s.d. (n=6 mice for each type of cells), representative from three independent experiments. (e) Representative FACS analysis of tdTomato⁺ cells stained with lineage markers (shown on the plots) in the BM of SCID mice transplanted with either SLRB/SLRH-HP cells and control BM cells (Ctr tdTomato⁺) at 12–14dpt. SLR factors were delivered in one polycistronic construct. Mice analysed: n=6 for each type of cells. Ery, Erythroid; Meg, Megakaryocytes. These data are representative of three independent experiments.