Necdin is dispensable for fetal hematopoiesis. (A) The frequency of immunophenotypic HSCs in necdin-null (Ndn-null) fetal livers (Lin⁻Sca-1⁺Mac1⁺CD48⁻CD150⁺ cells) quantified by flow cytometry was normal. The mean percentage (± SD) of Lin⁻Sca-1⁺Mac1⁺CD48⁻CD150⁺ cells in the fetal liver are shown (P = .64, n = 15). (B) Serial replating studies were performed using fetal liver cells. CFUs were quantified by methylcellulose culture using wild-type (WT) and necdin-null fetal liver cells. The methylcellulose cultures were serially replated weekly for 7 weeks. Mean values (± SD) are shown (n = 4). (C) Wild-type and necdin-null Lin⁻Sca1⁺Mac1⁺CD150⁺ cells (5 × 10²) were cultured on MS5 stromal cells for 4 weeks and tested for colony formation in the long-term culture-initiating cell assay. Data shown are the mean relative number of colonies formed relative to wild-type colonies (± SD) from 2 independent studies (P = .025, n = 5). (D) Cell-cycle analysis of Lin⁻Sca-1⁺Mac1⁺CD48⁻CD150⁺ cells was performed by staining with Hoechst 33342 and Ki67 and analyzed by flow cytometry using an FITC mouse IgG1 Ab as the isotype control. Ki67⁻ cells are defined as HSCs in G₀ (left panels). Data shown are the mean values (± SD; P = .5, n = 15, right panel). (E) Apoptosis of wild-type and necdin-null fetal liver HSCs was assessed using DAPI and annexin V staining. Data shown are the mean percentage (± SD) of annexin V⁺/DAPI⁻ Lin⁻Sca-1⁺Mac1⁺CD48⁻CD150⁺ cells (P = .71, n = 7). (F) Wild-type or necdin-null fetal liver cells (5 × 10⁵) were transplanted into lethally irradiated recipients, and spleen colonies were scored on days 8 and 12 after transplantation. Numbers indicate average colony numbers (± SD). CFU-spleen day 8 (CFU-S8), P = .53, n = 5; CFU-spleen day 12 (CFU-S12), P = .86, n = 5.