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.

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

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.

Quantitative label-free mass spectrometry reveals content and signaling differences between neonatal and adult platelets.

BACKGROUND: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. OBJECTIVES: This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. METHODS: We isolated resting umbilical cord blood-derived platelets from healthy full-term neonates (n = 8) and resting blood platelets from healthy adults (n = 6) and compared protein and phosphoprotein contents using data-independent acquisition mass spectrometry. RESULTS: We identified 4770 platelet proteins with high confidence across all samples. Adult and neonatal platelets were clustered separately by principal component analysis. Adult platelets were significantly enriched in immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched in ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched in phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched in phosphorylated proteins involved in insulin growth factor signaling. CONCLUSION: Using label-free data-independent acquisition mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation between neonatal and adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

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.

Phosphoproteomics reveals content and signaling differences between neonatal and adult platelets.

Background and Objective: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. Methods: We isolated resting umbilical cord blood-derived platelets from healthy full term neonates (n=9) and resting blood platelets from healthy adults (n=7), and compared protein and phosphoprotein contents using data independent acquisition mass spectrometry. Results: We identified 4745 platelet proteins with high confidence across all samples. Adult and neonatal platelets clustered separately by principal component analysis. Adult platelets were significantly enriched for immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched for ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched for phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched for phosphorylated proteins involved in insulin growth factor signaling. Conclusions: Using state-of-the-art mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation compared with adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of a molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

Neonatal Thrombocytopenia as a Presenting Finding in de novo Pyruvate Kinase Deficiency.

Thrombocytopenia is a common laboratory abnormality encountered in critically ill neonates. The broad differential for thrombocytopenia, and its association with potentially severe neonatal pathology, often presents a diagnostic dilemma prompting extensive evaluation. Hemolysis due to red cell enzymopathies is a rare cause of neonatal thrombocytopenia that is typically brief and self-limiting. Here, we present a case of thrombocytopenia, refractory to transfusion, associated with anemia and hyperbilirubinemia in a neonate with pyruvate kinase deficiency (PKD) arising from compound heterozygous PKLR mutations. The nature of the thrombocytopenia in this patient created considerable diagnostic uncertainty, which was ultimately resolved by whole-exome sequencing. This case emphasizes that inherited red cell defects, such as PKD, are important to consider in cases of neonatal thrombocytopenia.

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.

Genetically influenced tobacco and alcohol use behaviors impact erythroid trait variation.

Genome wide association studies (GWAS) have associated thousands of loci with quantitative human blood trait variation. Blood trait associated loci and related genes may regulate blood cell-intrinsic biological processes, or alternatively impact blood cell development and function via systemic factors and disease processes. Clinical observations linking behaviors like tobacco or alcohol use with altered blood traits can be subject to bias, and these trait relationships have not been systematically explored at the genetic level. Using a Mendelian randomization (MR) framework, we confirmed causal effects of smoking and drinking that were largely confined to the erythroid lineage. Using multivariable MR and causal mediation analyses, we confirmed that an increased genetic predisposition to smoke tobacco was associated with increased alcohol intake, indirectly decreasing red blood cell count and related erythroid traits. These findings demonstrate a novel role for genetically influenced behaviors in determining human blood traits, revealing opportunities to dissect related pathways and mechanisms that influence 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.

Mendelian randomization analyses clarify the effects of height on cardiovascular diseases.

An inverse correlation between stature and risk of coronary artery disease (CAD) has been observed in several epidemiologic studies, and recent Mendelian randomization (MR) experiments have suggested causal association. However, the extent to which the effect estimated by MR can be explained by established cardiovascular risk factors is unclear, with a recent report suggesting that lung function traits could fully explain the height-CAD effect. To clarify this relationship, we utilized a well-powered set of genetic instruments for human stature, comprising >1,800 genetic variants for height and CAD. In univariable analysis, we confirmed that a one standard deviation decrease in height (~6.5 cm) was associated with a 12.0% increase in the risk of CAD, consistent with previous reports. In multivariable analysis accounting for effects from up to 12 established risk factors, we observed a >3-fold attenuation in the causal effect of height on CAD susceptibility (3.7%, p = 0.02). However, multivariable analyses demonstrated independent effects of height on other cardiovascular traits beyond CAD, consistent with epidemiologic associations and univariable MR experiments. In contrast with published reports, we observed minimal effects of lung function traits on CAD risk in our analyses, indicating that these traits are unlikely to explain the residual association between height and CAD risk. In sum, these results suggest the impact of height on CAD risk beyond previously established cardiovascular risk factors is minimal and not explained by lung function measures.

TSABL: Trait Specific Annotation Based Locus predictor.

BACKGROUND: The majority of Genome Wide Associate Study (GWAS) loci fall in the non-coding genome, making causal variants difficult to identify and study. We hypothesized that the regulatory features underlying causal variants are biologically specific, identifiable from data, and that the regulatory architecture that influences one trait is distinct compared to biologically unrelated traits. RESULTS: To better characterize and identify these variants, we used publicly available GWAS loci and genomic annotations to build 17 Trait Specific Annotation Based Locus (TSABL) predictors to identify differences between GWAS loci associated with different phenotypic trait groups. We used a penalized binomial logistic regression model to select trait relevant annotations and tested all models on a holdout set of loci not used for training in any trait. We were able to successfully build models for autoimmune, electrocardiogram, lipid, platelet, red blood cell, and white blood cell trait groups. We used these models both to prioritize variants in existing loci and to identify new genomic regions of interest. CONCLUSIONS: We found that TSABL models identified biologically relevant regulatory features, and anticipate their future use to enhance the design and interpretation of genetic studies.

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.

High rate of extreme thrombocytosis indicates bone marrow hyperactivity and splenic dysfunction among congenital diaphragmatic hernia patients.

Pediatric extreme thrombocytosis (EXT, platelet count > 1000 x 103/µL) is rare. In a single center retrospective analysis of hospitalized children with EXT, infants with congenital diaphragmatic hernia (CDH) were overrepresented. In general pediatric patients, EXT is usually secondary to infection or inflammation, but most of the 14 CDH patients with EXT had no identifiable inciting factor. Instead, there was evidence that splenic dysfunction and bone marrow hyperactivity underlied EXT in CDH patients. None were associated with bleeding or thrombosis. Our findings identify mechanisms underlying EXT, and aid clinical interpretation and management of EXT in the pediatric population.

Thrombocytosis in an infant with a TRPV4 mutation: a case report.

Mutations in the calcium channel gene Transient Receptor Potential cation channel subfamily V member 4 (TRPV4) cause autosomal dominant skeletal dysplasia, with phenotypes ranging from mild to perinatal lethality. A recent report detailed enhanced proplatelet formation and increased murine platelet count in the context of TRPV4 activation. No prior reports have described platelet count abnormalities in human TRPV4 disease. Here, we report a case of prolonged thrombocytosis in the context of TRPV4-associated metatropic dysplasia that was lethal in the infantile period.

Airway emergency management in a pediatric hospital before and during the COVID-19 pandemic.

OBJECTIVE: Children's hospitals frequently care for infants with various life-threatening airway anomalies. Management of these infants can be challenging given unique airway anatomy and potential malformations. Airway emergency management must be immediate and precise, often demanding specialized equipment and/or expertise. We developed a Neonatal-Infant Emergency Airway Program to improve medical responses, communication, equipment usage and outcomes for all infants requiring emergent airway interventions in our neonatal and infant intensive care unit (NICU). PATIENTS AND METHODS: All patients admitted to our quaternary NICU from 2008 to 2019 were included in this study. Our program consisted of a multidisciplinary airway response team, pager system, and emergency equipment cart. Respiratory therapists present at each emergency event recorded specialist response times, equipment utilization, and outcomes. A multidisciplinary oversite committee reviewed each incident. RESULTS: Since 2008, there were 159 airway emergency events in our NICU (~12 per year). Mean specialist response times decreased from 5.9 ± 4.9 min (2008-2012, mean ± SD) to 4.3 ± 2.2 min (2016-2019, p = 0.12), and the number of incidents with response times >5 min decreased from 28.8 ± 17.8% (2008-2012) to 9.3 ± 11.4% (2016-2019, p = 0.04 by linear regression). As our program became more standardized, we noted better equipment availability and subspecialist communication. Few emergency situations (n = 9, 6%) required operating room management. There were 3 patient deaths (2%). CONCLUSIONS: Our airway safety program, including readily available specialists and equipment, facilitated effective resolution of airway emergencies in our NICU and multidisciplinary involvement enabled rapid and effective changes in response to COVID-19 regulations. A similar program could be implemented in other centers.

Airway emergency management in a pediatric hospital before and during the COVID-19 pandemic.

Objective: Children's hospitals frequently care for infants with various life-threatening airway anomalies. Management of these infants can be challenging given unique airway anatomy and potential malformations. Airway emergency management must be immediate and precise, often demanding specialized equipment and/or expertise. We developed a Neonatal-Infant Airway Safety Program to improve medical responses, communication, equipment usage and outcomes for infants requiring emergent airway interventions. Patients and Methods: All patients admitted to our quaternary neonatal and infant intensive care unit (NICU) from 2008-2019 were included in this study. Our program consisted of a multidisciplinary airway response team, pager system, and emergency equipment cart. Respiratory therapists present at each emergency event recorded specialist response times, equipment utilization, and outcomes. A multidisciplinary oversite committee reviewed each incident. Results: Since 2008, there were 159 airway emergency events in our NICU (~12 per year). Mean specialist response times decreased from 5.9±4.9 min (2008-2012, mean±SD) to 4.3±2.2 min (2016-2019, p=0.12), and the number of incidents with response times >5 min decreased from 28.8±17.8% (2008-2012) to 9.3±11.4% (2016-2019, p=0.04 by linear regression). As our program became more standardized, we noted better equipment availability and subspecialist communication. Few emergency situations (n=9, 6%) required operating room management. There were 3 patient deaths (2%). Conclusions: Our airway safety program, including readily available specialists and equipment, facilitated effective resolution of airway emergencies in our NICU and multidisciplinary involvement enabled rapid and effective changes in response to COVID-19 regulations. A similar program could be implemented in other centers.

Extreme thrombocytosis is associated with critical illness and young age, but not increased thrombotic risk, in hospitalized pediatric patients.

BACKGROUND: Extreme thrombocytosis (EXT, platelet count > 1000 × 103 /µL) is an uncommon but potentially clinically significant finding. Primary EXT in the setting of myeloproliferative disorders is linked to thrombotic and/or bleeding complications more frequently than secondary EXT, which typically occurs in reaction to infection, inflammation, or iron deficiency. However, comorbidities have been reported in adults with secondary EXT. Clinical implications of EXT in children are not well defined, as prior studies targeted small and/or specialized pediatric populations. OBJECTIVES: Our objectives were to determine etiologies and sequelae of EXT in a hospitalized general pediatric patient population. PATIENTS AND METHODS: We retrospectively analyzed EXT cases from a single-center pediatric cohort of ~80 000 patients over 8 years. RESULTS: Virtually all cases (99.8%) were secondary in nature, and most were multifactorial. Many cases of EXT occurred in children under 2 years old (47%) and/or during critical illness (55%). No thrombotic or bleeding events directly resulted from EXT, confirming a paucity of clinical complications associated with EXT in pediatric patients. There were indications that neonatal hematopoiesis and individual genetic variation influenced some cases, in addition to certain diagnoses (eg, sickle cell anemia) and clinical contexts (eg, asplenia). CONCLUSION: Our findings confirm that thrombotic events related to EXT are rare in pediatric patients, which can inform the use of empiric anti-platelet therapy.

Genetic determinants of increased body mass index mediate the effect of smoking on increased risk for type 2 diabetes but not coronary artery disease.

Clinical observations have linked tobacco smoking with increased type 2 diabetes risk. Mendelian randomization analysis has recently suggested smoking may be a causal risk factor for type 2 diabetes. However, this association could be mediated by additional risk factors correlated with smoking behavior, which have not been investigated. We hypothesized that body mass index (BMI) could help to explain the association between smoking and diabetes risk. First, we confirmed that genetic determinants of smoking initiation increased risk for type 2 diabetes (OR 1.21, 95% CI: 1.15-1.27, P = 1 × 10-12) and coronary artery disease (CAD; OR 1.21, 95% CI: 1.16-1.26, P = 2 × 10-20). Additionally, 2-fold increased smoking risk was positively associated with increased BMI (~0.8 kg/m2, 95% CI: 0.54-0.98 kg/m2, P = 1.8 × 10-11). Multivariable Mendelian randomization analyses showed that BMI accounted for nearly all the risk smoking exerted on type 2 diabetes (OR 1.06, 95% CI: 1.01-1.11, P = 0.03). In contrast, the independent effect of smoking on increased CAD risk persisted (OR 1.12, 95% CI: 1.08-1.17, P = 3 × 10-8). Causal mediation analyses agreed with these estimates. Furthermore, analysis using individual-level data from the Million Veteran Program independently replicated the association of smoking behavior with CAD (OR 1.24, 95% CI: 1.12-1.37, P = 2 × 10-5), but not type 2 diabetes (OR 0.98, 95% CI: 0.89-1.08, P = 0.69), after controlling for BMI. Our findings support a model whereby genetic determinants of smoking increase type 2 diabetes risk indirectly through their relationship with obesity. Smokers should be advised to stop smoking to limit type 2 diabetes and CAD risk. Therapeutic efforts should consider pathophysiology relating smoking and obesity.