Trisomy 21 (T21) is associated with baseline erythrocytosis, thrombocytopenia, neutrophilia, transient abnormal myelopoiesis (TAM), and myeloid leukemia of Down syndrome (ML-DS). TAM and ML-DS blasts harbor mutations in GATA1, resulting in the exclusive expression of the truncated isoform GATA1s. Germline GATA1s mutations in individuals without T21 cause congenital cytopenias, typically without a leukemic predisposition. To dissect the developmental effects of T21 and GATA1s, we used a combination of isogenic human induced pluripotent stem cells, primary human fetal and neonatal cells, and single-cell transcriptomics to interrogate hematopoietic progenitors differing only by chromosome 21 and/or GATA1 status. Both T21 and GATA1s induced early lineage skewing, and trajectory analysis revealed that GATA1s altered the temporal regulation of lineage-specific transcriptional programs, disrupting cell proliferation and maturation irrespective of chromosomal context. These studies uncovered unexpected heterogeneity and lineage priming in early, multipotent hematopoietic progenitors and identified transcriptional and functional maturation blocks linked to GATA1s.

BACKGROUND:Neonates with congenital anomalies frequently require perioperative allogeneic red blood cell (RBC) transfusion. Whole cord blood for autologous transfusion to neonates may provide an alternative RBC source, but whether sufficient volumes can be collected after delayed cord clamping to reduce allogeneic RBC requirements is unknown. STUDY DESIGN AND METHODS:Inclusion criteria were mothers delivering a viable infant >34 weeks' gestation. Sterile cord blood collection from the umbilical cord was performed at delivery as per routine obstetric indications. During storage at 4°C, we performed weekly blood gases. Blood culture, complete blood count, and hemolysis tests were performed at baseline and day 21. We compared the whole cord blood volume collected with each infant's allogeneic transfusion requirements. RESULTS:54 collection attempts yielded 49 collections with a mean volume of 54.1 mL (±20.3) after median delayed cord clamping of 46 seconds (IQR 12.0, 60.0). Among 39 blood cultures obtained, 3 grew organisms after vaginal delivery (3/27, 11.0% vs. 0/12, 0% cesarean delivery, p = .54). Hemolysis was stable during storage (baseline vs. day 21, median [IQR], 0.7% [0.4%-0.9%] vs. 0.7% [0.6%-1.1%], p = .08). CONCLUSIONS:Whole cord blood collection following delayed cord clamping was feasible, with volumes equal to 16.7 mL/kg, or one transfusion. Hemolysis was low, and although potassium increased during storage, it was consistent with patterns observed with adult donor stored whole blood. There were no positive blood cultures from collections during cesarean deliveries. Studies are needed to determine whether whole cord blood transfusions improve patient outcomes.

Down syndrome, or Trisomy 21 (T21), is uniquely associated with transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS). Both TAM and ML-DS harbor mutations in the erythro-megakaryocytic transcription factor GATA1 that lead to exclusive expression of GATA1s, a truncated isoform lacking the amino terminus. Consistent with the megakaryocytic phenotype and gene signature of TAM and ML-DS, we found that hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) with T21 and GATA1s display absent erythropoiesis but hyperproliferation of megakaryocytes and cytokine independence. Single-cell RNA sequencing of iPSC-derived hematopoietic progenitor cells (HPCs) revealed that T21 and GATA1s drive early commitment to the megakaryocyte lineage and block maturation, recapitulating a leukemic phenotype. To investigate the mechanisms underlying these diseases, we combined bulk RNA sequencing, ATAC-seq (assay for transposase-accessible chromatin with sequencing), and CUT&RUN (cleavage under targets and release using nuclease) for GATA1 in T21 HPCs expressing wtGATA1 vs. GATA1s. Multipotent CD41+235+ HPCs were differentiated and flow-purified from isogenic T21/wtGATA1 and T21/GATA1s iPSCs. RNA sequencing of these HPCs identified 1,292 differentially expressed genes (|log2FC| >0, adjusted p <0.05). Gene set enrichment analysis (GSEA) revealed enrichment of cell cycle pathways in T21/GATA1s HPCs, including G2/M checkpoint, E2F targets, and MYC targets. Parallel ATAC-seq identified 19,277 differentially accessible regions (adjusted p <0.05) in T21 HPCs expressing wtGATA1 vs. GATA1s. GSEA of differentially expressed genes with corresponding changes in chromatin accessibility again revealed positive enrichment of G2/M checkpoint genes, E2F targets, and MYC targets as well as negative enrichment of heme metabolism, consistent with the cytokine-independent hyperproliferation of megakaryocytes we observed. To determine whether GATA1s chromatin occupancy drives these transcriptional changes, we performed CUT&RUN for GATA1 in CD41+235+ HPCs and CD41+42b+ megakaryocytes differentiated from T21/wtGATA1 and T21/GATA1s iPSCs. In HPCs, GATA1s binding was globally decreased; compared to wtGATA1, 2,340 regions had decreased occupancy whereas only 62 regions had increased binding (false discovery rate ≤0.05). Genes with decreased GATA1s occupancy, chromatin accessibility, and expression in T21/GATA1s HPCs included CCND1 and CDKN1A, both of which inhibit cell cycle progression in G1 phase and play critical roles in the RB pathway. Notably, GATA1 itself plays an important role in this pathway through the GATA1-RB-E2F complex, which represses E2F to control cell proliferation, but GATA1s lacks the motif to form this complex. In stark contrast to the HPCs, GATA1s occupancy was globally increased in T21/GATA1s megakaryocytes (10,299 regions with increased occupancy compared to 279 regions with decreased occupancy; false discovery rate ≤0.05), including at both canonical megakaryocytic and erythroid loci. The essential megakaryocyte gene RUNX1 demonstrated increased chromatin accessibility and GATA1s occupancy, suggesting its role in the megakaryocyte bias and proliferation we observed with GATA1s. Taken together, our results suggest that GATA1s in T21 HPCs simultaneously promotes cell proliferation and megakaryopoiesis, yielding hyperproliferative, immature megakaryocytes due to absence of GATA1s binding in HPCs and inappropriate persistence of GATA1s at key loci in megakaryocytes. Our findings recapitulate the hallmarks of malignant transformation and offer mechanistic insights into the progression from T21 to TAM and ML-DS.

Trisomy 21 (T21) is associated with hematopoietic abnormalities including polycythemia, thrombocytopenia, transient abnormal myelopoiesis (TAM), and myeloid leukemia of Down syndrome (ML-DS). GATA1s, the truncated isoform of GATA1, is required for TAM and ML-DS pathogenesis, but causes cytopenias without leukemia in the absence of T21. To investigate how T21 and GATA1s each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or GATA1 status.HPCs were differentiated from 4 isogenic human iPSC lines: T21/wild-type GATA1 (WT), T21/GATA1s, euploid/WT, and euploid/GATA1s. CD41+235+ multipotent HPCs on Day 7 (D7) and lineage-biased HPCs on Days 9 (D9) and 11 (D11) were collected for scRNA-seq. Cluster annotation identified HPCs, megakaryocyte (MK)- or erythroid-biased HPCs, and erythroid, MK, and myeloid cells. Although GATA1s impairs erythropoiesis, T21/GATA1s HPCs showed an early erythroid bias that diminished by D11; euploid/GATA1s cells had strong MK and myeloid skews throughout. Both T21/WT and euploid/WT cells differentiated into all 3 lineages.Trajectory analysis revealed that T21 and GATA1s each impact lineage maturation. GATA1s delayed maturation of both euploid and T21 MKs. T21/GATA1s erythroid cells were less mature than T21/WT; there were insufficient euploid/GATA1s erythroid cells for analysis. GATA1s in T21 myeloid cells delayed maturation, but in euploid cells had no consistent effect across the 3 timepoints. T21/WT MKs and myeloid cells also appeared to be less mature than their euploid counterparts. Analysis of lineage-specific driver genes showed inappropriate expression levels with T21 or GATA1s relative to euploid/WT, suggesting that the impaired maturation is due to dysregulation of transcriptional programs.

Trisomy 21 (T21), or Down Syndrome (DS), is a common chromosomal disorder resulting from a third copy of chromosome 21 (HSA21). Transient myeloproliferative disorder (TMD) is a pre-leukemic condition that occurs only in neonates with DS and is characterized by a mutation in the transcription factor GATA1 that results in a truncated protein (GATA1s). We generated a pair of isogenic T21 lines derived from a patient with TMD that differ only in GATA1 status. The iPSC lines were characterized for pluripotency, differentiation potential, and genomic stability. These lines are a valuable resource for studying T21 hematopoietic diseases.

Introduction Trisomy 21 (T21) is associated with baseline erythrocytosis and thrombocytopenia and risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia of Down syndrome (ML-DS). TAM and ML-DS are characterized by mutations in the transcription factor GATA1, resulting in the truncated isoform GATA1s (G1S). We previously found that T21 increases erythropoiesis, while G1S severely impairs erythroid development but enhances megakaryocyte (MK) proliferation (Byrska-Bishop et al, JCI 2015). To better understand how T21 and G1S each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of multipotent and late hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or G1S status. Methods Four isogenic human induced pluripotent stem cell (iPSC) lines differing only by chromosome 21 (euploid vs. T21) and/or GATA1 (WT vs. G1S) status underwent hematopoietic differentiation by embryoid body formation. Cells were collected at 3 timepoints: on day 7 (D7), CD41 +235 + multipotent HPCs were purified by flow cytometry and on days 9 (D9) and 11 (D11), late HPCs biased to a single lineage were collected. Cells were sequenced by scRNA-seq and underwent quality control followed by clustering and analysis using Seurat. Clusters were manually annotated based on expression of 141 key hematopoietic genes, including aggregated expression of lineage markers. Early vs. committed lineage clusters were determined based on the relative expression of lineage-specific markers. Within each lineage, differentially expressed genes (DEGs) associated with a genetic background were assessed using the Wilcoxon rank-sum test. The χ² test was used to compare categorical data. A 2-tailed p or adjusted p <0.05 was considered statistically significant. Results Cluster annotation revealed HPC, early and committed erythroid and MK, and myeloid cells (Figure 1). D7 T21/G1S HPCs surprisingly showed an erythroid bias, with upregulation of genes such as GYPA and AHSP, while euploid/G1S HPCs showed a MK bias, with upregulation of genes including PF4V1 and PF4. With wtGATA1, neither euploid nor T21 D7 HPCs showed a significant lineage skew. On D9 and D11, the euploid/WT, T21/WT, and T21/G1S cells developed subpopulations committing to the erythroid lineage, while the euploid/G1S cells demonstrated minimal erythroid potential (Figure 1). Across all 3 timepoints, early and committed erythrocytes from T21 and euploid cells with G1S had a greater number of combined DEGs than their wtGATA1 counterparts (G1S vs. WT, T21: 692 vs. 378; euploid: 1235 vs. 599). On D9, T21 was associated with a greater erythroid skew (38.6% of T21/WT and 32.2% of T21/G1S cells vs. 27.8% of euploid/WT cells; p <0.00001). By D11, this erythroid drive was no longer apparent, and the T21/G1S erythrocytes were significantly less mature (Table 1; p <0.00001). These data suggest that T21 initially enhances erythropoietic drive, while G1S results in a near-complete erythroid block in euploid cells but an incomplete developmental block with T21. All 4 genotypes yielded MK subpopulations on D9 and D11 (Figure 1). With wtGATA1, euploid and T21 MKs were predominantly early MKs by D11 (Table 1). Interestingly, G1S was associated with enhanced MK commitment and maturation in the euploid context but arrest at the early stage with T21 (Table 1; p <0.00001). G1S was again associated with increased DEGs compared to wtGATA1 (G1S vs. WT, T21: 594 vs. 454; euploid: 1689 vs. 1310). These data suggest that G1S in a euploid background promotes MK differentiation, but T21 and G1S cooperate to generate immature, abnormal MKs. Conclusions Although the hematopoietic abnormalities and leukemic risk of T21 have been well-described, the underlying developmental events remain unclear. This scRNA-seq timecourse of HPCs differentiated from isogenic iPSCs reveals the individual and synergistic effects of T21 and G1S. Overall, T21 enhances erythropoietic drive while G1S alone suppresses erythropoiesis. G1S in euploid cells enhances megakaryopoiesis, but with T21, megakaryopoietic commitment and maturation are uncoupled with a strong bias to early MKs. Together, the competing effects of T21 and G1S appear to result in abnormal, immature erythrocytes and MKs with impaired lineage commitment, consistent with a pro-leukemic phenotype.

Transient myeloproliferative disorder (TMD) is a pre-leukemic condition that occurs only in neonates with Trisomy 21 (T21), and is attributed to a genetic interaction between the third copy of chromosome 21 (HSA21) and a mutation in the transcription factor GATA1 that results in a truncated protein (GATA1s). We generated a euploid iPSC line with a GATA1s mutation that is isogenic to a previously published pair of T21 lines with and without a GATA1 mutation. The line was characterized for pluripotency, differentiation potential, and genomic stability. This line is a valuable isogenic control for studying the T21 hematopoietic phenotype.

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator required for gene silencing during development. Although PRC2 is a well-established RNA-binding complex, the biological function of PRC2-RNA interaction has been controversial. Here, we study the gene-regulatory role of the inhibitory PRC2-RNA interactions. We report a nuclear long non-coding RNA, LEVER , which mapped 236 kb upstream of the β-globin cluster as confirmed by Nanopore sequencing. LEVER RNA interacts with PRC2 in its nascent form, and this prevents the accumulation of the H3K27 repressive histone marks within LEVER locus. Interestingly, the accessible LEVER chromatin, in turn, suppresses the chromatin interactions between the ε-globin locus and β-globin locus control region (LCR), resulting in a repressive effect on ε-globin gene expression. Our findings validate that the nascent RNA-PRC2 interaction inhibits local PRC2 function in situ. More importantly, we demonstrate that such a local process can in turn regulate the expression of neighboring genes.

The human hematopoietic stem cell harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, hematopoietic stem cells in the fetal liver undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. Here, we profile 26,407 fetal liver cells at both the transcriptional and protein level including ~7,000 highly enriched and functional fetal liver hematopoietic stem cells to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression reveals a generalizable signature defining functional fetal liver hematopoietic stem cells and allows for the stratification of enrichment strategies with high translational potential. More precisely, our integrated analysis identifies CD201 (endothelial protein C receptor (EPCR), encoded by PROCR ) as a marker that can specifically enrich for engraftment potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers. As such, it serves as a useful resource for the field and forms the basis for further biological exploration of strategies to retain the engraftment potential of hematopoietic stem cells ex vivo or induce this potential during in vitro hematopoietic stem cell generation.

INTRODUCTION:Sickle cell anemia is a mendelian disease that is noted for the heterogeneity of its clinical expression. Because of this, providing an accurate prognosis has been a longtime quest. AREAS COVERED:Reviewed are the benefits and shortcomings of testing for the major modulators of the severity of disease, like fetal hemoglobin and α thalassemia, along with studies that have attempted to link genetic variation with sub-phenotypes of disease in a predictive fashion. Induced pluripotent stem cells driven to differentiate into erythroid precursor cells provide another area for potential patient-specific drug testing. EXPERT OPINION:Fetal hemoglobin is the strongest modulator of sickle cell anemia but simply measuring its blood levels is an insufficient means of forecasting an individual's prognosis. A more precise method would be to know the distribution of fetal hemoglobin levels across the population of red cells, an assay not yet available. Prognostic measures have been developed using genetic and other signatures, but their predictive value is suboptimal. Widely applicable assays must be developed to allow a tailored approach to using the several new treatments that are likely to be available in the near future.