Perturbation of fetal liver hematopoietic stem and progenitor cell development by trisomy 21

Abstract

The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid-primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo- and lymphopoiesis.

Fig. 1.

Perturbation of T21 FL HSC/progenitor frequency. (A) Representative plots from normal FL (n = 13) and T21 FL (n = 8) CD34⁺ cells gated on CD34⁺CD38⁺CD19⁻ cells for CMP, MEP, and GMP, and on CD34⁺CD38^lo/− cells for HSC, MPP, and LMPP. (B) Mean + SEM HSC and progenitor frequencies in normal (n = 13; light bars) and T21 (n = 8) FL (dark bars) showing increased HSC and MEP frequency and reduced CBP and GMP. **P < 0.01; *P < 0.05.

Fig. 2.

Increased clonogenicity and megakaryocyte/erythroid potential of normal and T21 FL HSC and progenitors. (A) Clonogenicity of flow-sorted HSC and progenitors from normal (n = 8) and T21 (n = 5) FL (mean + SEM). Cells (100 cells/mL) were plated in Methocult H4230 with IL-3, IL-6, IL-11, SCF, FLT3, GM-CSF, TPO, and EPO. Clonogenicity of T21 HSC, CMP, and MEP was increased compared to normal FL. **P < 0.01; *P < 0.05. (B) Lineage read out of clonogenic data in A showing increased MK and MK-erythroid (MkE) colonies from T21 FL HSC, MPP, CMP, and MEP, and Blast-My colonies from T21 FL LMPP compared to normal FL. T21 HSC generated increased absolute numbers of CFU-MK, MkE, BFU-E, Blast-E, and Blast-My compared to normal FL HSC and T21 CMP and MEP increased CFU-MK, MkE, and Blast-E (for quantitation see Table S1). (C) Representative colonies (Scale bars, 100 μm.) and (D) colony cytospins (Scale bars, 10 μm.) from normal (Left) and T21 (Right) FL clonogenic assays. (E) Only Blast-My and Blast-E had secondary replating activity. No tertiary replating was seen. There was no difference between replating activity of normal and T21 FL Blast-My or Blast-E.

Fig. 3.

Impaired lymphoid differentiation of T21 FL HSC, LMPP, and ELP. (A) Representative plots from normal (Left; n = 11) and T21 (Right; n = 6) FL showing reduced Pre-proB (CD34⁺CD19⁺CD10⁻) and ProB progenitors (CD34⁺CD19⁺CD10⁺). (B) Mean B-lymphoid progenitor frequencies in normal (light bars; n = 11) and T21 (n = 6) FL (dark bars) showing reduced PreProB and ProB progenitors in T21 FL. ***P < 0.001; *P < 0.02. (C) Representative plots showing reduced CD34⁻CD19⁺ cells in T21 compared to normal FL and summary data in normal (light bars, n = 5) and T21 FL (dark bars, n = 8). *P < 0.05. (D) Mean number of CD20⁺ cells/high-power fields from normal (n = 4; light bars) and T21 FL (n = 4; dark bars). (E) Differentiation of flow-sorted 100 HSC, LMPP, and ELP on MS5 stroma showing representative results on day 0 and day 14 (day 7 also shown for ELP) from normal (n = 3) and T21 FL (n = 4) and (Right) mean absolute number of CD19⁺ cells on day 14 from normal FL HSC, LMPP, and ELP (light bars) or T21 HSC, LMPP or ELP (dark bars).

Fig. 4.

(A–D) Altered gene expression in T21 FL HSC/progenitors Mean gene expression levels by quantitative RT-PCR from flow-sorted HSC/progenitors (50 cells in triplicate for each population) from normal (n = 5; light bars) and T21 FL (n = 3; dark bars) shown relative to GAPDH. Significant differences between T21 and normal FL are shown as *P < 0.05; **P < 0.01, and ***P < 0.001, using Bayesian analysis of differences in mean (see SI Experimental Procedures).