Loss of necdin has no effects on adult HSC self-renewal in vivo. (A) Serial replating studies. CFUs were quantified by methylcellulose culture using BM mononuclear cells from mice reconstituted with wild-type (WT) or necdin-null (Ndn null) fetal liver cells. The methylcellulose cultures were serially replated weekly for 4 weeks. Mean values (± SD) are shown (n = 4). (B) Donor repopulation after serial BM transplantation. Initially, we transplanted 1 × 10⁶ fetal liver cells from wild-type or necdin-null mice (CD45.2) into lethally irradiated B6.SJL mice (CD45.1). Sixteen weeks after transplantation, we harvested 2 × 10⁶ BM cells from mice reconstituted with wild-type and necdin-null fetal liver cells and transplanted the cells into lethally irradiated B6.SJL mice (CD45.1). The frequency of donor-derived cells (CD45.2) in the peripheral blood was measured 16 weeks after primary, secondary, and tertiary transplantation by flow cytometry. No differences were found between the groups (n = 10). (C) Lethally irradiated recipient mice (CD45.1) were transplanted with 1 × 10⁶ wild-type or necdin-null fetal liver cells (CD45.2) plus 1 × 10⁶ competitor fetal liver cells (CD45.1). The graph shows the mean percentage (± SD) of donor-derived (CD45.2) cells in the peripheral blood 16 weeks after transplantation (n = 7, P = .50). (D) BM cells from mice reconstituted with wild-type or necdin-null fetal liver cells were stained for HSC surface markers and assessed for apoptosis using DAPI and annexin V staining. Data shown are mean percentage of annexin V⁺/DAPI⁻ HSCs (Lin⁻Sca1⁺Mac1⁺CD48⁻CD150⁺ cells) ± SD (P = .86, n = 4, left panel). The right panels show representative flow cytometry data: annexin V staining versus DAPI staining. (E-F) Fetal liver cell homing and lodging ability in irradiated and nonirradiated recipient mice was analyzed. Wild-type or necdin-null fetal liver cells (5 × 10⁶; CD45.2) were injected into lethally irradiated (E) or nonirradiated (F) recipient mice (CD45.1). BM cells were harvested 18 hours after injection and CD45.2⁺ donor-derived cells were enumerated on the Lin⁻Sca1⁺Mac1⁺ population by flow cytometry. Data shown are mean percentages (± SD) of cells that homed (E) or lodged (F) within the Lin⁻Sca1⁺Mac1⁺ cells (E: P = .92, F: P = .48, n = 5).