Figure 3.

c-Kit Deficiency Promotes Expansion of Hematopoietic Stem Cells from GFG-Derived iPSCs

(A) Strategy to induce HSCs from iPSCs using c-Kit deficient host mice. iPSCs were injected subcutaneously and Dox administered for 4 weeks to induce GFG expression (as in Figure 1B). Host mice were then administered polyI:C at weeks 14–16. At 20 weeks, WBMCs from teratoma-bearing mice were transplanted into irradiated mice, and PB/BM chimerism tracked. Secondary transplantations were performed at 16 weeks after the primary transplantation.

(B) Representative flow cytometric plots for CD45.1 and CD45.2 expression in the PB from teratoma-bearing C57BL/6 and c-Kit-deficient mice after 4–6 weeks poly(I:C) administration.

(C) Percentage CD45.1⁺ chimerism in the PB (left) and BM (right). Data are the means ± SD from two independent experiments (n = 6). ^∗∗p < 0.005, ^∗p < 0.05.

(D) Percentage of (GFG iPSC-derived) CD45.1⁺ PB chimerism of in primary and secondary recipient mice (n = 5).

(E and F) Colony potential of 2.0 × 10⁴ host CD45.2⁺ and GFG-derived CD45.1⁺ BM cells of secondary recipients, after 11-day culture in Methocult. Total number of colonies displayed in (E) and colony type displayed in (F). Data are means ± SD from two independent experiments (n = 3). Colony cells were morphologically identified as neutrophils (n), macrophages (m), erythroblasts (E) and megakaryocytes (M). ^∗∗p < 0.005.

(G) Representative flow cytometric plots and gating for the Kit⁺Sca1⁺Lineage⁻ fraction within the BM of secondary recipient mice transplanted with GFG teratoma-derived blood cells 24 weeks post-transplantation. Lin⁻, lineage negative cells; KSL, Lin⁻Kit⁺Sca1⁺ cells.