Fig. 3: HSC potential is restricted to CD31⁺CD45⁺c-Kit⁺CD41⁻ embryo explant cells and requires SCF and IL-3.

a Experimental schematic. Embryos isolated from E8.5 (5–7sp) concepti were cultured as in Fig. 1b, dissociated and fractionated by FACS based on CD31, CD45, and c-Kit cell surface expression and then transplanted at four ee/recipient into lethally irradiated CD45.1 + CD45.2 + recipients along with 2 × 10⁵ CD45.1⁺ WBM cells. b Representative flow cytometry plots denoting sorted and transplanted populations (A: CD31⁺CD45⁺c-Kit⁺CD41⁺; B: CD31⁺CD45⁺ c-Kit⁺CD41⁻; C: CD31⁺CD45⁺c-Kit⁻). c %CD45.2⁺ PB of recipients at four (light-colored circles) and 16 weeks (dark-colored circles) post-transplant. Each circle represents an individual recipient. Five independent experiments are depicted. In each experiment all sorted cells for each population were transplanted into one separate recipient. Engraftment was only detected upon transplantation of population B. d Experimental schematic. E8.5 (5–7sp) YS-Ex and Em-Ex were cultured at the air-liquid interface for 9–10 days in different combinations of SCF, FLT3L and IL-3 and either analyzed by flow cytometry or transplanted to test for dHSC. e Cultured explants were analyzed by flow cytometry for CD31⁺CD45⁺ and CD31⁺CD45⁺c-Kit⁺CD41⁻cells after 5 (n = 2, with the exception of Em-Ex SCF + IL-3 + FLT3L + , Em-Ex SCF + IL-3 + and Em-Ex IL-3 + FLT3L + where n = 3) or 10 days of culture (n = 3, with the exception of Em-Ex SCF + IL-3 + FLT3L + , Em-Ex SCF + IL-3 + and Em-Ex IL-3 + FLT3L + where n = 4). Each circle represents an individual culture. Bars stand for average. Results from four independent experiments are shown. Left panel: Em-Ex-explants. Right panel: YS-Ex. Statistical differences with the SCF + Flt3l + IL-3 + control are indicated: (*p < 0.05 and **p < 0.01, two-sample t-test). f CD45.2⁺ Em-Ex were transplanted into lethally irradiated CD45.1⁺CD45.2⁺ recipients at four ee/recipient along with 2 × 10⁵ CD45.1⁺ WBM cells. %CD45.2⁺ PB four (gray circles) and 16 weeks (black circles) post-transplant is shown. Each circle represents an individual recipient. Cumulative data from 13 independent experiments. The majority of the analyzed conditions yielded an engraftment that was marginally statistically higher than the No-Growth Factor condition (**p < 0.01, *p < 0.05, #p < 0.1; eWrs-test and two-sample t-test). Horizontal bars indicate averages. Error bars denote standard deviation. Source data are provided as a Source Data file