Clonogenic assays, liquid cultures, and gene expression of CD34⁺ cells from DS and normal FL. (A-C) CD34⁺ cells purified from freshly isolated MNCs from DS (n = 5) and gestation-matched normal (n = 4) FLs were plated at 500 to 1000 cells/well with interleukin-3 (IL-3), IL-6, IL-11, stem cell factor, Flt3 ligand, granulocyte-macrophage colony-stimulating factor, Tpo, and Epo and counted after 12 to 14 days. (A) Cloning efficiency of DS-FL CD34⁺ cells was higher than normal FL CD34⁺ cells (78 ± 7 vs 15 ± 3). (B) Absolute numbers of all myeloid progenitors were increased in DS-FL compared with normal FL: erythroid (198 + 2 vs 30 + 5 colonies/5 × 10² cells; P < .001), megakaryocyte/megakaryocyte-erythroid (60 + 6 vs 5 + 2.6 colonies/5 × 10² CD34⁺ cells; P < .001), CFU-GEMM (57 + 4 vs 10 + 0.5; P < .001), and CFU-G/GM/M (98 + 6 vs 29 + 1; P < .001). (C) Serial replating of individual CFU-GEMM from DS-FL (n = 3) showed markedly increased replating efficiency compared with normal FL CFU-GEMM (n = 3): secondary replating of normal FL CFU-GEMM produced only occasional CFU-e with no tertiary replating ability, whereas secondary replating of DS-FL CFU-GEMM produced tertiary CFU-GEMM and erythroid colonies (BFU-e and CFU-e) and tertiary replating of DS-FL secondary CFU-GEMM gave rise to CFU-e, which had no replating ability. (D) Erythroid liquid cultures: MACS-purified DS-FL (n = 5) and normal gestation-matched FL CD34⁺ cells (n = 3) cultured with Epo, stem cell factor, Flt3 ligand, and IL-3 generated similar numbers of GlyA⁺ cells after 7 and 10 days of culture (inset: flow cytometry of total cells after 10 days of culture). (E) Megakaryocyte liquid cultures: MACS-purified DS-FL CD34⁺ cells (n = 5) cultured with Tpo generated a slightly higher total number of cells compared with normal FL CD34⁺ cells (n = 3) after 10 days (P = .07) with a similar proportion of CD61⁺ cells and morphologically normal megakaryocytes (inset: flow cytometry of total cells and cytospin of megakaryocytes after 10 days of culture). (F) Expression of full-length GATA1, truncated GATA1 (GATA1s), RUNX1, and GATA2 transcripts by FL CD34⁺ cells in T21 (n = 3) and normal (n = 3) FL was measured by Taqman quantitative reverse-transcribed polymerase chain reaction in 3 independent experiments, each comparing a T21 with a normal sample. Expression levels in each of the T21 samples normalized against a normal FL sample is shown.