Hepatoblasts and KITL alone are sufficient to expand FL LSK. (A) Image of a spheroid at day 6 (D6) of culture (upper image, brightfield) and representative single-stack IF of a D6 spheroid of a co-culture of E14.5 hepatoblasts and CD45⁺ FL cells; DNA (blue), E-Cadh (green), and CD45 (white). (B) Numbers of LSK cells recovered from spheroids after 2 (D2) and 4 (D4) days of culture. Cultures with three populations: hepatoblasts (CD31⁻Gp38⁻PDGFRα⁻EpCAM⁻E-Cadh⁺ cells), mesenchymal cells (CD31⁻Gp38⁻PDGFRα⁺ cells), and MCs (CD31⁻PDGFRα⁻Gp38⁺). ACK2 antibody was added on D0 of CD45⁺ cells co-cultured with hepatoblasts. (C) Numbers of CD45⁺ cells and Lin⁻Kit⁺Sca-1⁻ (LK) cells isolated from individual spheroids (n = 3). All bar plots indicate average ± SD. n indicates the number of independent biological samples analyzed. Statistical analysis was calculated using two-way ANOVA, followed by Tukey’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.