GATA1 in Normal and Pathologic Megakaryopoiesis and Platelet Development.

GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.

Single-cell transcriptomics reveal synergistic and antagonistic effects of T21 and GATA1s on hematopoiesis.

Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, and transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform of GATA1 , while germline GATA1s mutations in a non-T21 context lead to congenital cytopenias without a leukemic predisposition. This suggests that T21 and GATA1s perturb hematopoiesis independently and synergistically, but this interaction has been challenging to study in part due to limited human cell and murine models. To dissect the developmental impacts of GATA1s on hematopoiesis in euploid and T21 cells, we performed a single-cell RNA-sequencing timecourse on hematopoietic progenitors (HPCs) derived from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/or GATA1 status. These HPCs were surprisingly heterogeneous and displayed spontaneous lineage skew apparently dictated by T21 and/or GATA1s. In euploid cells, GATA1s nearly eliminated erythropoiesis, impaired MK maturation, and promoted an immature myelopoiesis, while in T21 cells, GATA1s appeared to compete with the enhanced erythropoiesis and suppressed megakaryopoiesis driven by T21 to give rise to immature erythrocytes, MKs, and myeloid cells. T21 and GATA1s both disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. These findings in an isogenic system can thus be attributed specifically to T21 and GATA1s and suggest that these genetic changes together enhance HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.

Machine learning to optimize automated RH genotyping using whole-exome sequencing data.

ABSTRACT: Rh phenotype matching reduces but does not eliminate alloimmunization in patients with sickle cell disease (SCD) due to RH genetic diversity that is not distinguishable by serological typing. RH genotype matching can potentially mitigate Rh alloimmunization but comprehensive and accessible genotyping methods are needed. We developed RHtyper as an automated algorithm to predict RH genotypes using whole-genome sequencing (WGS) data with high accuracy. Here, we adapted RHtyper for whole-exome sequencing (WES) data, which are more affordable but challenged by uneven sequencing coverage and exacerbated sequencing read misalignment, resulting in uncertain predictions for (1) RHD zygosity and hybrid alleles, (2) RHCE∗C vs. RHCE∗c alleles, (3) RHD c.1136C>T zygosity, and (4) RHCE c.48G>C zygosity. We optimized RHtyper to accurately predict RHD and RHCE genotypes using WES data by leveraging machine learning models and improved the concordance of WES with WGS predictions from 90.8% to 97.2% for RHD and 96.3% to 98.2% for RHCE among 396 patients in the Sickle Cell Clinical Research and Intervention Program. In a second validation cohort of 3030 cancer survivors (15.2% Black or African Americans) from the St. Jude Lifetime Cohort Study, the optimized RHtyper reached concordance rates between WES and WGS predications to 96.3% for RHD and 94.6% for RHCE. Machine learning improved the accuracy of RH predication using WES data. RHtyper has the potential, once implemented, to provide a precision medicine-based approach to facilitate RH genotype-matched transfusion and improve transfusion safety for patients with SCD. This study used data from clinical trials registered at ClinicalTrials.gov as #NCT02098863 and NCT00760656.

Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells.

ABSTRACT: During development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FL-derived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FL- and iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FL- and iPSC-derived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.

Synergistic roles of DYRK1A and GATA1 in trisomy 21 megakaryopoiesis.

Patients with Down syndrome (DS), or trisomy 21 (T21), are at increased risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (ML-DS). Both TAM and ML-DS require prenatal somatic mutations in GATA1, resulting in the truncated isoform GATA1s. The mechanism by which individual chromosome 21 (HSA21) genes synergize with GATA1s for leukemic transformation is challenging to study, in part due to limited human cell models with wild-type GATA1 (wtGATA1) or GATA1s. HSA21-encoded DYRK1A is overexpressed in ML-DS and may be a therapeutic target. To determine how DYRK1A influences hematopoiesis in concert with GATA1s, we used gene editing to disrupt all 3 alleles of DYRK1A in isogenic T21 induced pluripotent stem cells (iPSCs) with and without the GATA1s mutation. Unexpectedly, hematopoietic differentiation revealed that DYRK1A loss combined with GATA1s leads to increased megakaryocyte proliferation and decreased maturation. This proliferative phenotype was associated with upregulation of D-type cyclins and hyperphosphorylation of Rb to allow E2F release and derepression of its downstream targets. Notably, DYRK1A loss had no effect in T21 iPSCs or megakaryocytes with wtGATA1. These surprising results suggest that DYRK1A and GATA1 may synergistically restrain megakaryocyte proliferation in T21 and that DYRK1A inhibition may not be a therapeutic option for GATA1s-associated leukemias.

Generation of CHOPi-008-B, a euploid iPSC line from a patient with Trisomy 21 and a GATA1 mutation.

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.

Tropomyosin 1 deficiency facilitates cell state transitions to enhance hemogenic endothelial cell specification during hematopoiesis.

Tropomyosins coat actin filaments and impact actin-related signaling and cell morphogenesis. Genome-wide association studies have linked Tropomyosin 1 (TPM1) with human blood trait variation. Prior work suggested that TPM1 regulated blood cell formation in vitro, but it was unclear how or when TPM1 affected hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC) model systems, TPM1 knockout was found to augment developmental cell state transitions, as well as TNFα and GTPase signaling pathways, to promote hemogenic endothelial (HE) cell specification and hematopoietic progenitor cell (HPC) production. Single-cell analyses showed decreased TPM1 expression during human HE specification, suggesting that TPM1 regulated in vivo hematopoiesis via similar mechanisms. Indeed, analyses of a TPM1 gene trap mouse model showed that TPM1 deficiency enhanced the formation of HE during embryogenesis. These findings illuminate novel effects of TPM1 on developmental hematopoiesis.

Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

In vivo hematopoietic stem cell modification by mRNA delivery.

Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual's lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP-messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti-human CD117/LNP-based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT.

Generation of 2 isogenic clones from a patient with Trisomy 21 and a GATA1 mutation.

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.

HIC2 controls developmental hemoglobin switching by repressing BCL11A transcription.

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal ß-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer-promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A.

Body mass index and adipose distribution have opposing genetic impacts on human blood traits.

Body mass index (BMI), hyperlipidemia, and truncal adipose distribution concordantly elevate cardiovascular disease risks, but have unknown genetic effects on blood trait variation. Using Mendelian randomization, we define unexpectedly opposing roles for increased BMI and truncal adipose distribution on blood traits. Elevated genetically determined BMI and lipid levels decreased hemoglobin and hematocrit levels, consistent with clinical observations associating obesity and anemia. We found that lipid-related effects were confined to erythroid traits. In contrast, BMI affected multiple blood lineages, indicating broad effects on hematopoiesis. Increased truncal adipose distribution opposed BMI effects, increasing hemoglobin and blood cell counts across lineages. Conditional analyses indicated genes, pathways, and cell types responsible for these effects, including Leptin Receptor and other blood cell-extrinsic factors in adipocytes and endothelium that regulate hematopoietic stem and progenitor cell biology. Our findings identify novel roles for obesity on hematopoiesis, including a previously underappreciated role for genetically determined adipose distribution in determining blood cell formation and function.

Transfusion and Cellular Therapy in Pediatric Sickle Cell Disease.

Red blood cell (RBC) transfusion is critical in managing acute and chronic complications of sickle cell disease. Alloimmunization and iron overload remain significant complications of transfusion therapy and are minimized with prophylactic Rh and K antigen RBC matching and iron chelation. Matched sibling donor hematopoietic stem cell transplant (HSCT) is a curative therapeutic option. Autologous hematopoietic stem cell (HSC)-based gene therapy has recently shown great promise, for which obtaining sufficient HSCs is essential for success. This article discusses RBC transfusion indications and complications, transfusion support during HSCT, and HSC mobilization and collection for autologous HSCT with gene therapy.

VPS4A Mutations in Humans Cause Syndromic Congenital Dyserythropoietic Anemia due to Cytokinesis and Trafficking Defects.

The Congenital Dyserythropoietic Anemia (CDA) Registry was established with the goal to facilitate investigations of natural history, biology, and molecular pathogenetic mechanisms of CDA. Three unrelated individuals enrolled in the registry had a syndrome characterized by CDA and severe neurodevelopmental delay. They were found to have missense mutations in VPS4A, a gene coding for an ATPase that regulates the ESCRT-III machinery in a variety of cellular processes including cell division, endosomal vesicle trafficking, and viral budding. Bone marrow studies showed binucleated erythroblasts and erythroblasts with cytoplasmic bridges indicating abnormal cytokinesis and abscission. Circulating red blood cells were found to retain transferrin receptor (CD71) in their membrane, demonstrating that VPS4A is critical for normal reticulocyte maturation. Using proband-derived induced pluripotent stem cells (iPSCs), we have successfully modeled the hematologic aspects of this syndrome in vitro, recapitulating their dyserythropoietic phenotype. Our findings demonstrate that VPS4A mutations cause cytokinesis and trafficking defects leading to a human disease with detrimental effects to erythropoiesis and neurodevelopment.

Mechanistic and Translational Advances Using iPSC-Derived Blood Cells.

Human induced pluripotent stem cell (iPSC)-based model systems can be used to produce blood cells for the study of both hematologic and non-hematologic disorders. This commentary discusses recent advances that have utilized iPSC-derived red blood cells, megakaryocytes, myeloid cells, and lymphoid cells to model hematopoietic disorders. In addition, we review recent studies that have defined how microglial cells differentiated from iPSC-derived monocytes impact neurodegenerative disease. Related translational insights highlight the utility of iPSC models for studying pathologic anemia, bleeding, thrombosis, autoimmunity, immunodeficiency, blood cancers, and neurodegenerative disease such as Alzheimer's.

2019 sickle cell disease guidelines by the American Society of Hematology: methodology, challenges, and innovations.

The American Society of Hematology (ASH) convened 5 guideline panels to develop clinical practice recommendations addressing 5 management areas of highest importance to individuals living with sickle cell disease: pain, cerebrovascular complications, pulmonary and kidney complications, transfusion, and hematopoietic stem cell transplant. Panels were multidisciplinary and consisted of patient representatives, content experts, and methodologists. The Mayo Clinic Evidence-Based Practice Center conducted systematic reviews based on a priori selected questions. In this exposition, we describe the process used by ASH, including the GRADE approach (Grades of Recommendations, Assessment, Development and Evaluation) for rating certainty of the evidence and the GRADE Evidence to Decision Framework. We also describe several unique challenges faced by the guideline panels and the specific innovations and solutions used to address them, including a curriculum to train patients to engage in guideline development, dealing with the opioid crisis, and working with indirect and noncomparative evidence.

Mutation-specific signaling profiles and kinase inhibitor sensitivities of juvenile myelomonocytic leukemia revealed by induced pluripotent stem cells.

Juvenile myelomonocytic leukemia (JMML) is an uncommon myeloproliferative neoplasm driven by Ras pathway mutations and hyperactive Ras/MAPK signaling. Outcomes for many children with JMML remain dismal with current standard-of-care cytoreductive chemotherapy and hematopoietic stem cell transplantation. We used patient-derived induced pluripotent stem cells (iPSCs) to characterize the signaling profiles and potential therapeutic vulnerabilities of PTPN11-mutant and CBL-mutant JMML. We assessed whether MEK, JAK, and PI3K/mTOR kinase inhibitors (i) could inhibit myeloproliferation and aberrant signaling in iPSC-derived hematopoietic progenitors with PTPN11 E76K or CBL Y371H mutations. We detected constitutive Ras/MAPK and PI3K/mTOR signaling in PTPN11 and CBL iPSC-derived myeloid cells. Activated signaling and growth of PTPN11 iPSCs were preferentially inhibited in vitro by the MEKi PD0325901 and trametinib. Conversely, JAK/STAT signaling was selectively activated in CBL iPSCs and abrogated by the JAKi momelotinib and ruxolitinib. The PI3Kδi idelalisib and mTORi rapamycin inhibited signaling and myeloproliferation in both PTPN11 and CBL iPSCs. These findings demonstrate differential sensitivity of PTPN11 iPSCs to MEKi and of CBL iPSCs to JAKi, but similar sensitivity to PI3Ki and mTORi. Clinical investigation of mutation-specific kinase inhibitor therapies in children with JMML may be warranted.

Testicular endothelial cells are a critical population in the germline stem cell niche.

Maintenance of adult tissues depends on stem cell self-renewal in local niches. Spermatogonial stem cells (SSC) are germline adult stem cells necessary for spermatogenesis and fertility. We show that testicular endothelial cells (TECs) are part of the SSC niche producing glial cell line-derived neurotrophic factor (GDNF) and other factors to support human and mouse SSCs in long-term culture. We demonstrate that FGF-2 binding to FGFR1 on TECs activates the calcineurin pathway to produce GDNF. Comparison of the TEC secretome to lung and liver endothelial cells identified 5 factors sufficient for long-term maintenance of human and mouse SSC colonies in feeder-free cultures. Male cancer survivors after chemotherapy are often infertile since SSCs are highly susceptible to cytotoxic injury. Transplantation of TECs alone restores spermatogenesis in mice after chemotherapy-induced depletion of SSCs. Identifying TECs as a niche population necessary for SSC self-renewal may facilitate fertility preservation for prepubertal boys diagnosed with cancer.

Generation of a human Juvenile myelomonocytic leukemia iPSC line, CHOPi001-A, with a mutation in CBL.

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disorder of early childhood characterized by expansion of clonal myelomonocytic cells and hyperactive Ras/MAPK signaling. The disorder is caused by somatic and/or germline mutations in genes involved in the Ras/MAPK and JAK/STAT signaling pathways, including CBL. Here we describe the generation of an iPSC line with a homozygous CBL c.1111T->C (Y371H) mutation, designated CHOPJMML1854.