Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells

Abstract

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryonic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage.

Fig. 1.

Isolation and characterization of isogenic disomic and trisomic clones. (A) Overview depicting subcloning of disomic and trisomic isogenic iPS cells from a mixed culture of cells. (B) Subcloned isogenic disomic and trisomic clones express Oct4, Tra1-60, and exhibit stable karyotypes lacking chromosomal abnormalities other than trisomy 21. (Magnification: 20×.)

Fig. 2.

Fetal-like definitive hematopoietic differentiation via EBs. (A) Schematic detailing the differentiation of human pluripotent cells into hematopoietic lineages via EBs. (B) Globin expression in cells isolated from day 10 EBs and CFU-E colonies relative to GAPDH. CD34⁺ bone marrow and CD34⁺ FL represent positive control samples from differentiated CD34⁺ bone marrow and FL cells, respectively. N.D., not detected.

Fig. 3.

Hematopoietic populations of in vitro differentiated disomic and trisomic pluripotent cells. (A) CD31/KDR⁺ cells derived from day 8 EBs. (B and C) CD43 and CD235⁺ populations isolated from day 10 and day 11 EBs, respectively. Error bars represent SEM, and P values were determined by the Student t test; **P < 0.005.

Fig. 4.

Colony-forming potential of differentiated pluripotent cells. Colonies formed by isogenic (A), ES (B), and independent iPS (C) in methylcellulose colony-forming assay. (D) Megakaryocyte colonies formed by isogenic cells. Error bars represent SEM, and P values were determined by the Student t test; *P < 0.05, **P < 0.005.