System to study the effect of trisomy 21 expression on DS-related pathologies. a Schematic of the inducible XIST RNA-mediated silencing system in Down syndrome iPSCs, in which XIST induces formation of a condensed, heterochromatic chromosome 21 “Barr Body”. b Neural stem cell (rosette) formation after 15 days neural differentiation, with and without induced expression to silence one chromosome 21. The samples treated with dox have significantly more neural stem cells at this time point. Scale bar: 100 μm. c Quantification of the number of neural rosettes at day 15 for two of the isogenic XIST-transgenic subclones. The iPS cells used were from a male, but because regulatory counting elements are removed from the XIST cDNA, this system is also compatible with female cells. Error bars represent s.e.m. from three independent experiments

Trisomy silencing during hematopoietic differentiation. a Schematic of hematopoietic differentiation from DS iPSCs. b Association of H3K27me3 heterochromatic mark with XIST RNA in day 9 purified CD34⁺ differentiated cells treated with dox. Quantification shows over 90% of cells have H3K27me3 marker associated with XIST paints. c RNA FISH of APP and XIST on day 9 differentiating cells. APP RNA transcription foci from all three alleles in untreated cells (top). In treated cells (bottom), only two APP transcription foci are detected, indicating transcriptional silencing induced by XIST expression. Quantification shows over 90% of XIST expressing CD34⁺ cells have only two APP transcription foci (which are not from XIST expressing chromosome). Error bars for b and c represent s.e.m. from three independent scorings

Effect of XIST-induced trisomy silencing on colony-forming potential of DS iPSCs. Colony-forming potential of multiple isogenic transgenic DS iPS lines with and without XIST-induced chromosome 21 silencing for a megakaryocytes, b erythrocytes, c granulocytes, and d monocytes. Experiments were repeated at least three times. Error bars represent s.e.m. from three independent experiments and P values were calculated by Student t test; *P < 0.05, **P < 0.01, ***P < 0.001

Impact of trisomy silencing on hematopoietic populations during differentiation. a A simplified schematic of steps in the hematopoietic differentiation process with markers used to define specific cell population. EHT: endothelial to hematopoietic transition. b Early hematopoietic progenitor populations detected at day 11 and 14 differentiation for clone 5. Quantifications are represented as the ratio of doxcycycline-treated cells to untreated cells. Transgenic cells, which XIST is induced have less CD43⁺ early hematopoietic progenitor cells at both day 11 and 14 of differentiation. c, d Hemogenic endothelium-like populations identified during hematopoietic differentiation for clone 5. Quantifications are represented as the ratio of treated cells to untreated cells. Chromosome 21 silencing does not affect the formation of hemogenic endothelium-like populations at day 8 of differentiation, using either of the two sets of markers to define this population. Error bars represent s.e.m. from five independent experiments and P values were calculated by Student t test; *P < 0.05, ***P < 0.001

Effects of trisomy silencing on day 14 purified CD43⁺ hematopoietic progenitors. a Colony-forming potential from equal numbers of purified day 14 differentiated CD43⁺ hematopoietic progenitor cells for (i) megakaryocytes, (ii) erythrocytes, (iii) granulocytes, and (iv) monocytes. b, c Gene expression analysis on b three IGF signaling-related genes and two chromosome 21 genes in purified day 14 CD43⁺ hematopoietic progenitors, and on c a panel of hematopoietic regulators, which are dynamically changing in different hematopoietic subpopulations. Expression levels were normalized to GAPDH and represented as the ratio of treated cells to untreated cells. Error bars represent s.e.m. from three independent experiments and P values were calculated by Student t test; *P < 0.05, **P < 0.01, ***P < 0.001

Trisomy 21 contributes to increased sensitivity of CD43⁺ hematopoietic progenitor production to IGF inhibition. a Inhibition of IGF signaling by 1 μM PPP, an IGF inhibitor, has distinct effects on the CD34⁺ and CD43⁺ populations. Production of CD43⁺ early hematopoietic progenitor populations are more sensitive to IGF inhibition, whereas CD34⁺CD43⁻ hemogenic endothelium-enriched population is only slightly affected. b Analysis of the effects of IGF inhibition at three lower concentrations identifies the window of sensitivity for the CD43⁺ cells. Drug/no drug ratios were calculated by the percentage of each population in treated cultures divided by the percentage of each population in untreated cultures. c Trisomy silencing reduces the sensitivity of CD43⁺ cell production to IGF signaling inhibition, suggesting greater reliance of trisomic CD43⁺ progenitors on IGF signaling. Drug/no drug ratios were calculated as in b and dox/no dox ratios were calculated by the drug/no drug ratio of dox-treated samples divided by drug/no drug ratio of samples without dox, to reflect the increased reliance on IGF signaling in trisomic CD43⁺ hematopoietic progenitors. Error bars represent s.e.m. from three independent experiments and P values were calculated by Student t test; **P < 0.01, ***P < 0.001

Schematic of how trisomy 21 affects distinct steps in hematopoiesis. Trisomy 21 initially increases the production of hematopoietic progenitor cells as they emerge from the endothelial-to-hematopoietic transition. A likely enhanced EHT process is accompanied by increased IGF signaling. Trisomy 21 also increases the colony-forming potential of these hematopoietic progenitor cells