Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cells

Abstract

Patients with Down syndrome (trisomy 21, T21) have hematologic abnormalities throughout life. Newborns frequently exhibit abnormal blood counts and a clonal preleukemia. Human T21 fetal livers contain expanded erythro-megakaryocytic precursors with enhanced proliferative capacity. The impact of T21 on the earliest stages of embryonic hematopoiesis is unknown and nearly impossible to examine in human subjects. We modeled T21 yolk sac hematopoiesis using human induced pluripotent stem cells (iPSCs). Blood progenitor populations generated from T21 iPSCs were present at normal frequency and proliferated normally. However, their developmental potential was altered with enhanced erythropoiesis and reduced myelopoiesis, but normal megakaryocyte production. These abnormalities overlap with those of T21 fetal livers, but also reflect important differences. Our studies show that T21 confers distinct developmental stage- and species-specific hematopoietic defects. More generally, we illustrate how iPSCs can provide insight into early stages of normal and pathological human development.

Fig. 1.

T21 iPSCs produce normal levels of early hematopoietic progenitors. (A) Schematic of hematopoietic differentiation protocol via EB formation with the following cytokines: BMP4, VEGF, SCF, TPO, FLT3, bFGF, EPO, IL-3, IL-11, and IGF1. (B) Photograph of iPSC-derived EB culture with hematopoietic cells released into the medium. Original magnification, 10×. (C) Flow cytometry analysis showing CD31⁺KDR⁺ hematoendothelial precursor cells (Left) and CD34^+/−43⁺235⁺41⁺ progenitors within EBs (Center), and released into the medium (Right). (D) Methylcellulose colony assays of various purified populations. Cytokines include SCF, IL-3, EPO, and GMCSF. Results show mean values ± SEM, n = 3 per group. (E) Globin gene expression in erythroid colonies from iPSCs or fetal liver (FL) determined by quantitative real-time PCR. Charts show fraction of α- (Left) or β-like (Right) genes. Ten to 20 colonies were pooled per sample, n = 3 per group. (F) Frequency of CD43⁺41⁺235⁺ progenitor cells in EB cultures on day 7–8 of hematopoietic differentiation (n = 14 and 11 independent experiments for euploid and T21 iPSCs, respectively. (G) Scatter plots of microarray data showing average mRNA expression values in purified CD43⁺41⁺235⁺ progenitors from between-group comparison of three euploid and three T21 biological replicate samples.

Fig. 2.

Propensity for erythroid differentiation by T21 iPSCs. (A) Flow cytometry analysis of suspension cells in day 12 differentiation cultures showing mature hematopoietic lineages: erythroid (Ery, CD41⁻235⁺), megakaryocytic (Meg, CD41⁺42⁺), and myeloid (CD45⁺18⁺). (B) May–Grunwald Giemsa-stained cells from EB suspension cultures at days 12 and 20. (Scale bars, 50 μm.) (C) Distribution of lineage-committed cells in EB suspension cultures at days 12–14 of differentiation. n = 3–5 independent experiments per iPSC line. (D) Summary of data with all iPSC lines combined according to genotype (n = 15 per group).

Fig. 3.

Increased erythroid progenitors in T21 iPSC differentiation cultures. CD43⁺41⁺235⁺ hematopoietic progenitors derived from three euploid and four T21 iPSC lines were analyzed. (A) Methylcellulose colony assays of CD43⁺41⁺235⁺ progenitors containing SCF, IL-3, EPO, GM-CSF, and (B) Colony-forming megakaryocyte (CFU-Mk) assays that include TPO, IL-3, and IL-6. Results show mean values ± SEM for three independent experiments per iPSC line. (C) Summary of data with all iPSC lines combined according to genotype for methylcellulose colony assays and (D) CFU-Mk assays. Results show mean values ± SEM, n = 9 for WT, n = 12 for T21. (E) Representative hematopoietic colonies from iPSC-derived progenitors. (Scale bars, 200 μm.) Meg, megakaryocyte. (F) Morphology of cells from iPSC-derived erythroid and myeloid colonies. May–Grunwald Giemsa stain. (Scale bars, 20 μm.)