Increased erythroid and megakaryocytic potential of trisomy 21 fetal hematopoietic cells. (A) Methylcellulose colony assays of mononuclear cells (MNCs) from trisomy 21 (T21, n = 8) and control (C, n = 8) fetal livers. The numbers of burst-forming unit–erythroid (BFU-E) colonies are normalized to the numbers of colony-forming unit–granulocyte macrophage (CFU-GM) colonies obtained from the same culture dishes. The numbers of colony-forming unit–erythroid (CFU-E) and colony-forming unit–megakaryocyte (CFU-Mk) colonies are normalized to the numbers of CFU-GM obtained from the same number of cells plated in parallel GM cultures (y-axis, CFU-E, BFU-E, or CFU-Mk:CFU-GM ratio). CFU-Mk formation was assessed in semisolid cultures that were subsequently dehydrated, fixed, and stained with anti-GPIIb/IIIa antibody; CFU-Mk were scored based on GPIIb/IIIa positively staining cells. Colony assays were performed in triplicate. Results are shown as mean values plus or minus SD. *P = .014, **P < .005, ***P = .021. (B) Representative examples of BFU-E colony morphology from T21 and C fetal liver cells. T21 BFU-E colonies tended to be larger, with the majority approximately twice the size of control colonies. Original magnification, 10X. Photographs were taken using a Carl Zeiss Axiovert 25 microscope (Carl Zeiss, Thornwood, NY) equipped with a digital color camera (Canon Powershot A640; Canon, Lake Success, NY) and Axiovision acquisition software (Zeiss). (C) Large CFU-Mk, defined as more than 50 cells per colony, generated from T21 (n = 8) and C (n = 8) fetal liver MNCs. The y-axis shows the ratio of large CFU-Mk normalized to total CFU-Mk colonies generated by the same fetal liver specimen. Results are shown as mean values plus or minus SD. *P = .028. (D) Growth of megakaryocytes in liquid culture. T21 (n = 4) and C (n = 3) fetal liver MNCs were seeded into serum-free growth medium supplemented with thrombopoietin (Tpo). The y-axis shows relative numbers of megakaryocytes, as defined by expression of the lineage marker CD41a. By 14 days, approximately 90% of the cells expressed megakaryocyte markers CD41a and CD42 (not shown). Results are indicated as mean values plus or minus SD. (E) Representative examples of proplatelet formation in T21 and C fetal liver liquid megakaryocyte cultures on day 13. Proplatelet formation appeared on day 8 of culture and increased with each subsequent day in both T21 and C liquid cultures. By visual inspection, there were no differences in the extent of proplatelet formation between T21 and C samples. Original magnification, 20X. Photographs were taken using an Olympus IX70 microscope (Olympus, Center Valley, PA) equipped with a Photometrics Coolsnap camera (Photometrics, Pleasanton, CA) and Openlab version 5.5 acquisition software (Improvision, Waltham, MA). (F) Hematopoietic development of T21 (n = 11) and C (n = 12) fetal liver MNCs 5 to 10 weeks after transplantation into NOD/SCID/IL-2Rγc^null mice. Mice that received T21 MNCs exhibited an increased fraction of erythroid cells (Glycophorin A⁺, left panel) and megakaryocytes (CD41a⁺, right panel) within the human hematopoietic compartment in bone marrow of chimeric mice. Results are shown as the mean values plus or minus SD. *P < .005, **P = .007.