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Red blood cell (RBC) transfusion is one of the most common medical treatments, with more than 10 million units transfused per year in the United States alone. Alloimmunization to foreign Rh proteins (RhD and RhCE) on donor RBCs remains a challenge for transfusion effectiveness and safety. Alloantibody production disproportionately affects patients with sickle cell disease who frequently receive blood transfusions and exhibit high genetic diversity in the Rh blood group system. With hundreds of RH variants now known, precise identification of Rh antibody targets is hampered by the lack of appropriate reagent RBCs with uncommon Rh antigen phenotypes. Using a combination of human-induced pluripotent stem cell (iPSC) reprogramming and gene editing, we designed a renewable source of cells with unique Rh profiles to facilitate the identification of complex Rh antibodies. We engineered a very rare Rh null iPSC line lacking both RHD and RHCE. By targeting the AAVS1 safe harbor locus in this Rh null background, any combination of RHD or RHCE complementary DNAs could be reintroduced to generate RBCs that express specific Rh antigens such as RhD alone (designated D--), Goa+, or DAK+. The RBCs derived from these iPSCs (iRBCs) are compatible with standard laboratory assays used worldwide and can determine the precise specificity of Rh antibodies in patient plasma. Rh-engineered iRBCs can provide a readily accessible diagnostic tool and guide future efforts to produce an alternative source of rare RBCs for alloimmunized patients.

Rh alloimmunization remains a challenge for patients with sickle cell disease despite transfusion of serologic Rh C, E and K antigen-matched red cells. Inheritance of altered RH alleles contributes to the prevalence of Rh antibodies following blood transfusion in patients with SCD and explains approximately one-third of cases. The remainder appear to be stimulated by altered Rh proteins on African-American donor red cells. Matching of patients with donors based on RH genotype may mitigate Rh alloimmunization, but the feasibility and resources required are not known. We compared RH allele frequencies between patients with SCD (n=857) and African-American donors (n=587) and show that RH allele frequencies are similar. Overall, 29% of RHD and 53% of RHCE alleles are altered in patients and African-American donors. We modeled RH genotype matching compared to serologic Rh D, C, and E, along with K antigen-matching, and found that approximately twice the number of African-American donors would be required for RH genotype versus Rh serologic matching at our institution. We demonstrate that African-American donor recruitment is necessary to maintain an adequate supply of C, E, and K negative donor units to avoid depleting the Rh negative (RhD-) blood supply. Our results suggest that prophylactic RH genetic matching for patients with SCD is feasible with a donor pool comprised primarily of African-Americans and would optimize utilization of our existing minority donor inventory. The current cost of RH genotyping all minority donors and management of the data remain limiting factors.

Introduction Genome wide association studies (GWAS) have catalogued thousands of loci that influence blood traits, but genetic mechanisms and impacted cell types are often unknown. While some regulatory factors are blood cell-autonomous, other extrinsic regulatory factors respond to systemic physiology. Epidemiologic studies have correlated generalized obesity (increased body mass index, BMI) and high cholesterol with anemia, but cell types and mechanisms that mediate these effects are incompletely understood, and these studies do not provide evidence for causality. Thus, we wanted to use genetic effects of BMI and cholesterol on hemoglobin level (HGB) and other blood traits to infer causal relationships, identify relevant cell types, and propose genetic mechanisms. Methods Mendelian randomization (MR), akin to a "genetic randomized controlled trial", uses variants linked to an exposure trait to estimate causal effects of that exposure on a given outcome. 1 Random variant allele allocation at meiosis enables this approach to address confounding and reverse causality that can otherwise preclude causal inference from epidemiologic and cohort studies. Further, effects of multiple factors can be parsed using multivariable MR and causal mediation analyses to help explain genetic associations. 2 Our study used gender- and age-adjusted GWAS summary statistics from European individuals. Results We hypothesized that increased genetically determined BMI would decrease HGB. Using a MR framework, we found that a 5 kg/m 2 (1 standard deviation unit) increase in BMI decreased hemoglobin by 0.06±0.01 g/dL (p=1x10 -5, Fig). Increased BMI also decreased erythrocyte count, and unexpectedly also decreased platelet and white blood cell counts (all with p Similar to BMI, a 1 SD unit increase in total cholesterol decreased HGB (0.10±0.03 g/dL, p=2x10 -3, Fig). However, genetic effects of cholesterol were restricted to erythroid traits (HGB and hematocrit). Multivariable and mediation analyses confirmed that effects of BMI and cholesterol functioned through distinct genetic mechanisms. We speculated that multilineage effects from BMI could reflect a genetic predisposition to accumulate bone marrow adipose tissue, which can impact HPCs and hematopoiesis. 3 A tendency for 'central' adiposity increases one's waist-hip ratio (WHR), and increases cardiovascular disease risk concordant with BMI. Unexpectedly, we found that increased WHR, in contrast to BMI, increased HGB (0.08±0.02 g/dL, p=9x10 -6) as well as erythrocyte, platelet, and white blood cell counts (all with p Conclusions Our results confirm that BMI and cholesterol negatively impact HGB at a genetic level, consistent with clinical observations. The unexpected multilineage effects of genetically determined BMI most likely reflects a tendency to accumulate bone marrow adipose tissue, which in turn impacts HPCs and downstream blood cell production. Our findings suggest that adjustment for BMI and adiposity traits may be considered in blood trait GWAS analyses and illuminate opportunities to functionally dissect related genes and molecular pathways. References 1. Hemani, G. et al. Elife 7, (2018). PMID: 29846171. 2. Sanderson, E. et al. Int. J. Epidemiol. 48, 713-727 (2019). PMID: 30535378. 3. Wang, H. et al. Front. Endocrinol.. 9, 694 (2018). PMID: 30546345. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Despite transfusion of Rh matched red cells for patients with sickle cell disease, Rh alloimmunization remains a persistent challenge. Rh specificities can be complex, resulting from RH genetic diversity found in patients and donors. Antibody identification is hampered by the lack of appropriate reagent red cells, especially those that can identify antibodies against high prevalence or low prevalence Rh antigens. We used human induced pluripotent stem cells (iPSCs) with the goal of producing renewable red cell reagents to both screen for Rh alloimmunization and to aid complex antibody identification. Methods: We generated a panel of iPSCs that include Rh null, D--, lack the high prevalence antigens hr S or hr B, or express uncommon Rh antigens such as V, VS, Go a, or DAK. For the Rh null line, we used CRISPR/Cas9 genetic engineering to disrupt RHCE via a large deletion in a D- iPSC. For D--, RHD was inserted into the AAVS1 safe harbor locus of an Rh null iPSC line using zinc finger nucleases resulting in a line that constitutively expresses RhD but no RhCE. iPSCs with uncommon variants were reprogrammed from RH genotyped donors or engineered similar to the generation of the D-- line. Hematopoietic differentiation by embryoid body formation was used to generate hematopoietic progenitors that were subsequently cultured towards the erythroid lineage. Mature iRBCs were ficin treated and tested with patient plasma with previously identified Rh antibodies using gel agglutination assays. Results: Rh null iPSC-derived RBCs (iRBCs) showed complete absence of cell surface Rh protein by flow cytometry, while D-- iRBCs showed Rh protein expression levels comparable to D-ce+ iRBCs using an anti-D/CE antibody. We assessed RBC agglutination of Rh null, D--, hr S-, hr B-, VVS+, Go a+, and DAK+ iRBCs using standard Rh typing reagents (Ortho). The reprogrammed uncommon donor iRBCs agglutinated with monoclonal anti-Rh antibodies as predicted by RH genotype, while the Rh null iRBCs showed no agglutination with all 5 common Rh antibodies and D-- iRBCs showed agglutination with anti-D reagents only. Rh null iRBCs showed no agglutination against patient plasma containing anti-D, while D-- iRBCs agglutinated. While D- RHCE*ce homozygous iRBCs showed strong agglutination against patient plasma containing anti-hr S, Rh null, D--, and hr S- iRBCs did not agglutinate. No iRBCs showed agglutination by plasma containing anti-V/VS while VVS+ iRBCs showed strong agglutination. Similarly, no iRBCs showed agglutination by plasma containing anti-Go a while Go a+ iRBCs showed strong agglutination. Detection of most antibodies against Rhce on iRBCs was enhanced by ficin treatment whereas antibodies with D specificity did not require ficin treated cells for detection. Conclusion: We suggest that genetically engineered iPSCs expressing uncommon Rh antigen phenotypes that are difficult or impossible to obtain from red cell donors can expedite antibody identification. Rh null and D-- iRBCs could be useful to discriminate antibodies against RhD versus RhCE. Customized iPSCs that lack high prevalence or express low prevalence Rh antigens could potentially standardize antibody evaluation in patients with complex Rh specificities. Disclosures No relevant conflicts of interest to declare.

One of the oldest and most deeply studied problems in developmental gene expression is the switch from fetal to adult type hemoglobin production in red blood cell precursors. Interest in this question has been fueled by its relevance to genetic blood disorders such as sickle cell disease (SCD) and thalassemia. BCL11A is a transcriptional repressor that is thought to directly silence the fetal β-type globin (HBG1/2) genes in adult erythroid cells. Transcriptome and RNA polymerase II profiling indicate that the BCL11A gene is transcribed considerably more highly in adult erythroblasts compared to fetal cells, accounting in large part for corresponding changes in BCL11A protein levels. Yet, the mechanism governing BCL11A developmental regulation is still unclear. To identify novel regulators of the fetal-to-adult globin switch, we interrogated our recent CRISPR based genetic screens that employed single guide RNAs (sgRNAs) targeting transcription factors (Huang et al., Blood, 2020) and uncovered HIC2, a penta-dactyl zinc finger DNA binding protein bearing a BTB/POZ domain as a novel regulator of hemoglobin switching. HIC2 is expressed more highly in fetal erythroblasts compared to adult cells, a pattern inverse to that of BCL11A. Overexpression (OE) of HIC2 in the adult type erythroid HUDEP2 cell line stimulated the expression of 322 genes while impairing that of 224 genes (FDR < 0.01 and fold change ≥ 2). The most highly upregulated genes (>150-fold) were HBG1/2. Upregulation was accompanied by gains in chromatin accessibility and histone H3K27acetylation of HBG1/2, and increased chromatin contacts between the distal globin gene enhancer (LCR) and the HBG1/2 genes. Overexpression of HIC2 in primary human erythroblasts also significantly increased HBG1/2 mRNA and protein levels, sufficient to reduce cell sickling in SCD patient-derived erythroid cells. HIC2 OE lowered BCL11A mature and pre-mRNA production, indicating that HIC2 attenuates BCL11A transcription. Forced expression of BCL11A restored HBG1/2 silencing in HIC2 OE HUDEP2 cells, suggesting that BCL11A repression accounts for the effects of HIC2 on fetal globin genes. ChIP-seq revealed a strong HIC2 binding peak at the erythroid BCL11A +55 enhancer. HIC2 OE reduced chromatin accessibility and H3K27acetylation of the +55 enhancer, as well as the enhancer-promoter contacts, suggesting that HIC2 directly decommissions the enhancer to attenuate BCL11A transcription. The BCL11A +55 enhancer contains two consensus HIC2 binding motifs under the HIC2 peak adjacent to GATA:E-box and GATA motifs. CRISPR-mediated mutagenesis of both HIC2 motifs raised BCL11A basal level transcription and diminished the ability of overexpressed HIC2 to repress BCL11A transcription. Notably, HIC2 OE impaired binding of transcription factor GATA1 at the +55 enhancer, suggesting that this enhancer is under developmental control. Indeed, GATA1 binding and chromatin accessibility of +55 enhancer were virtually undetectable in HUDEP1 cells, which represent a more fetal-like state. CRISPR-mediated depletion of HIC2 in HUDEP1 cells reversed this pattern with gains in GATA1 binding, chromatin accessibility, and BCL11A transcription. In sum, HIC2 emerges as a critical regulator of hemoglobin switching that operates by imposing developmental stage-specific control onto a BCL11A transcriptional enhancer. Disclosures Blobel: Fulcrum therapeutics: Consultancy; Pfizer: Research Funding.

Painful vaso-occlusive crisis (VOC) is the most common complication of sickle cell disease (SCD). Increasing evidence suggests that vaso-occlusion is initiated by increased adherence of sickle red blood cells (RBCs) to the vascular endothelium. Thus, the mechanisms that remove endothelial-attached sickle RBCs from the microvasculature are expected to be critical for optimal blood flow and prevention of VOC in SCD. We hypothesized that patrolling monocytes (PMos), which protect against vascular damage by scavenging cellular debris, could remove endothelial-adherent sickle RBCs and ameliorate VOC in SCD. We detected RBC (GPA+)-engulfed material in circulating PMos of patients with SCD, and their frequency was further increased during acute crisis. RBC uptake by PMos was specific to endothelial-attached sickle, but not control, RBCs and occurred mostly through ICAM-1, CD11a, and CD18. Heme oxygenase 1 induction, by counteracting the cytotoxic effects of engulfed RBC breakdown products, increased PMo viability. In addition, transfusions, by lowering sickle RBC uptake, improved PMo survival. Selective depletion of PMos in Townes sickle mice exacerbated vascular stasis and tissue damage, whereas treatment with muramyl dipeptide (NOD2 ligand), which increases PMo mass, reduced stasis and SCD associated organ damage. Altogether, these data demonstrate a novel mechanism for removal of endothelial attached sickle RBCs mediated by PMos that can protect against VOC pathogenesis, further supporting PMos as a promising therapeutic target in SCD VOC.

Children with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS.

Introduction RBC alloimmunization is common in patients with sickle cell disease (SCD). Despite serological matching RBCs for major Rh antigens, Rh alloimmunization remains problematic. The Rh blood group is encoded by two genes RHD and RHCE, which exhibit extensive nucleotide polymorphism and chromosome structural changes, resulting in the formation of Rh variant antigens. Rh variants can result in loss of protein epitopes or expression of neo-epitopes, and are common in SCD patients. Hence SCD patients harboring Rh variants can be predisposed to Rh alloimmunization. Given the limitation of traditional serologic antigen typing for detection of Rh variants, molecular genotyping has become required. A DNA microarray-based platform, BioArray RHCE and RHD BeadChip (Immuncor) is available for RH genotyping. However, it detects the most common, but not all, variants. Whole exome sequence data have been used for prediction of Rh variants (Chou, et. al, Blood Adv., 2017), offer some advantages, including detection of rare variants, structural rearrangements and copy number variation. However, whole genome sequence (WGS) analysis of RHD/RHCE is challenging due to difficulties in mapping next generation sequencing (NGS) reads to this duplicated gene family. We developed a computational algorithm to identify RH variants using WGS data. Methods The pipeline included three major components, RH allele database construction, RH variant calling, and classification of Rh blood group according the identified variants. The RH allele database was built based on NCBI Blood Group Antigen Gene Mutation (BGMUT) and International Society of Blood Transfusion (ISBT) database. Since the alleles in the BGMUT and ISBT databases were specified according to conventional RH genes (RHD, L08429; RHCE, DQ322275) that are different from those on reference human genome, we first called the variations based on the reference human genome. The positions of the identified variations were subsequently corrected to match with the BGMUT and ISBT annotation system. Next, the NGS reads with low base quality and/or mapping quality were discarded during the variation calling step. Synonymous and non-synonymous amino acid changes were characterized for each polymorphism. Haplotypes were constructed for the segments with NGS read support. Gene sequencing coverage was calculated to determine gene deletions or amplifications. Lastly, we implemented an algorithm to predict RH genotypes based on a selection of candidate alleles by read-mapping profile which considers both sequence variations and sequence consistency followed by a likelihood-based ranking of all pairwise combinations of the selected alleles. The allele combination with the highest likelihood is considered the most likely pair of alleles at a given locus. Patient specimens used in this study were from participants of the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer. 2018). Results We validated our method in a cohort of 58 SCD patients whose RH genotypes had been determined by BioArray RhCE and RhD BeadChip and supplementary molecular tests that identify the most common variants among individuals of African descent. In this validation cohort including a total of 11 RHD and 13 RHCE alleles, our approach achieved a concordance rate of 85.85% (91 of 106 alleles) for RHD and 83.02% (88 of 106 alleles) for RHCE genotyping. WGS was highly sensitive in distinguishing homozygosity from heterozygosity of genes. By comparing the numbers of NGS reads on RH regions and whole genome average coverage, heterozygous deletion can be determined. Since WGS provides comprehensive genotyping, our analysis identified single nucleotide polymorphisms that were not identified by the BeadChip and supplemental molecular testing. The final source of discordance was likely due to the short read length of NGS such that haplotype phases cannot be correctly predicted if the variations are separated by thousands of base pairs, for which long read DNA sequencing or RNA/cDNA sequencing are required. Evaluation of the identified discrepancies is ongoing. Conclusions We developed and validated a diagnostic method for RH genotyping that leveraged the accuracy and flexibility of RH genotyping based on WGS data. With further optimization of our method, this may be useful for RBC genotype matching sickle cell patients to blood donors in the future. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy.

Sickle cell disease (SCD) is characterized by hemolytic anemia and increased entrapment of sickle red blood cells (RBCs) via attachment to the underlying activated vascular endothelium, resulting in vaso-occlusive crisis (VOC), marked by severe pain. The endothelial scavenging patrolling monocytes (PMos) expressing high levels of the heme oxygenase 1 (HO-1), a heme degrading enzyme, were recently shown to protect against vaso-occlusion in SCD, although their ability to scavenge endothelial-attached sickle RBCs was not tested. Here, we found that circulating PMos from SCD patients showed roughly 5% ± 0.5% engulfed GPA+ or Band3+ RBC specific material as compared to 0.7% ± 0.04% in healthy donor (HD) PMos or 0.85% ± 0.07% in SCD classical monocytes (CMos) as detected by flow as well as imagining flow cytometry, suggesting that PMos uptake RBCs in SCD. To further investigate this, RBCs purified from HDs (to mimic transfused cells) or SCD patients were labelled with CFSE, and co-cultured with purified monocytes without or with human microvascular endothelial cells (HMVEC). We found 11% ± 0.5% CFSE+ PMos in co-cultures with SCD RBCs in presence of HMVEC as compared to 2.7% ± 0.4% when cultured with HD RBCs, indicating that PMos engulf sickle RBCs, but not HD RBCs. Low levels of CFSE+ PMos (2-3%) were detected in cocultures with either sickle or control RBCs in the absence of HMVEC, implicating that PMos preferentially uptake endothelial cell (EC)-attached sickle RBCs. In contrast to PMos, CMos always had minimal CFSE+ reactivity (2-3%) when cultured with sickle RBCs or HD RBCs with or without HMVEC, supporting a role for PMos, but not CMos, as scavengers of sickle RBCs in the vasculature. Further analysis revealed significantly increased (two-fold) levels of annexin V+ apoptotic marker on sickle RBC engulfed PMos (CFSE+PMos), but also higher levels of HO-1 ((50% ± 3%) as compared to non-engulfed (CFSE-) PMos (1.3% ± 0.4%), suggesting that induction of HO-1 upon uptake of sickle RBCs may counteract the cytotoxic effects of engulfed RBC breakdown products in PMos. To test this hypothesis, monocytes were pre-treated with tin protoporphyrin IX (SnPPIX) to inhibit HO-1 activity prior to coculture with sickle RBCs and HMVEC. We found increased apoptosis in PMos from SnPPIX-treated (18% ± 0.8%) as compared to untreated cultures (6.6% ± 0.6%), consistent with a cytoprotective role of HO-1 induction in sickle RBC-engulfed PMos. Antibody blocking studies identified ICAM-1, VCAM-1, CD11a, CD18 as well as CD16 as key molecules involved in PMo-HMVEC-sickle RBC interactions, but not CD11b, CD11c, CD31, PSGL-1, CD32, CD64, Fc a/m receptor and phosphatidylserine. Interestingly, HMVEC activation induced by heme treatment resulted in significantly higher CFSE+ PMos (17.8% ± 0.9%) when cultured with sickle RBCs, but not HD RBCs (3.5% ± 0.6%), indicating that PMos preferentially uptake sickle RBCs bound to heme-treated ECs. To formally test this, DiI labelled mouse sickle or control RBCs were transfused to heme-treated Nr4a1-GFP mice which express GFP on their circulating PMos, followed by perfusion to remove non-EC attached cells. Using confocal microscopy, we detected DiI+ sickle but not control non-sickle RBCs, engulfed by GFP+ PMos in the vasculature of perfused recipients, confirming uptake of EC-attached sickle RBCs by PMos. Consistent with increased EC-attached sickle RBCs during VOC, SCD patients experiencing acute VOC (n=12) showed increased frequency of GPA+ PMos (13% ± 0.1% vs 5% ± 0.2%, p Disclosures No relevant conflicts of interest to declare.

Acute megakaryoblastic leukemia (AMKL) is more frequently observed in Down syndrome (DS) patients, in whom it is often preceded by a transient myeloproliferative disorder (TMD). The development of DS-TMD and DS-AMKL requires not only the presence of the trisomy 21 but also that of GATA1 mutations. Despite extensive studies into the genetics of DS-AMKL, the importance of epigenetic deregulation in this disease has been unexplored. We performed DNA methylation profiling at different stages of development of DS-AMKL and analyzed the dynamics of the epigenetic program. Early genome-wide DNA methylation changes can be detected in trisomy 21 fetal liver mononuclear cells, prior to the acquisition of GATA1 mutations. These early changes are characterized by marked loss of DNA methylation at genes associated with developmental disorders, including those affecting the cardiovascular, neurological, and endocrine systems. This is followed by a second wave of changes detected in DS-TMD and DS-AMKL, characterized by gains of methylation. This new wave of hypermethylation targets a distinct set of genes involved in hematopoiesis and regulation of cell growth and proliferation. These findings indicate that the final epigenetic landscape of DS-AMKL is the result of sequential and opposing changes in DNA methylation occurring at specific times in the disease development.

Red blood cell (RBC) transfusion is a key treatment of patients with sickle cell disease (SCD) but remains complicated by RBC immunization. In the present study, we evaluated the effects of antigen matching for Rh D, C, and E, and K and transfusion from African American donors in 182 patients with SCD. Overall, 71 (58%) chronic and 9 (15%) episodically transfused patients were alloimmunized. Fifty-five (45%) chronic and 7 (12%) episodically transfused patients were Rh immunized. Of 146 antibodies identified, 91 were unexplained Rh antibodies, one-third of which were associated with laboratory evidence of delayed transfusion reactions. Fifty-six antibodies occurred in patients whose RBCs were phenotypically positive for the corresponding Rh antigen and 35 in patients whose RBCs lacked the antigen and were transfused with Rh-matched RBCs. High-resolution RH genotyping revealed variant alleles in 87% of individuals. These data describe the prevalence of Rh alloimmunization in patients with SCD transfused with phenotypic Rh-matched African American RBCs. Our results suggest that altered RH alleles in both the patients and in the donors contributed to Rh alloimmunization in this study. Whether RH genotyping of patients and minority donors will reduce Rh alloimmunization in SCD needs to be examined.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children initiated by mutations that deregulate cytokine receptor signaling. Studies of JMML are constrained by limited access to patient tissues. We generated induced pluripotent stem cells (iPSCs) from malignant cells of two JMML patients with somatic heterozygous p.E76K missense mutations in PTPN11, which encodes SHP-2, a nonreceptor tyrosine phosphatase. In vitro differentiation of JMML iPSCs produced myeloid cells with increased proliferative capacity, constitutive activation of granulocyte macrophage colony-stimulating factor (GM-CSF), and enhanced STAT5/ERK phosphorylation, similar to primary JMML cells from patients. Pharmacological inhibition of MEK kinase in iPSC-derived JMML cells reduced their GM-CSF independence, providing rationale for a potential targeted therapy. Our studies offer renewable sources of biologically relevant human cells in which to explore the pathophysiology and treatment of JMML. More generally, we illustrate the utility of iPSCs for in vitro modeling of a human malignancy.

Causing leukocyte activation and upregulation of adhesion molecules on endothelial cells. CD16+ monocytes, also known as endothelial patrolling monocytes, normally scavenge the damaged cells and debris from the vasculature. As compared to other monocyte subsets or immune cell types, the CD16+monocyte subset expresses higher levels of the anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme. Given the role of CD16+ monocytes as scavengers of debris on endothelial cells, we tested the hypothesis that this subset may protect SCD vasculature from the ongoing hemolytic insult through expression of high levels of HO-1. We found roughly 35% of circulating CD16+ monocytes from SCD patients expressed very high levels of HO-1 as compared to 5% in healthy controls. The HO-1hi SCD monocytes expressed significantly (30%) less TNF-a compared to HO-1lo monocytes following stimulation, consistent with anti-inflammatory effects of HO-1. We hypothesized that uptake of free hemoglobin/heme was responsible for high HO-1 expression levels in SCD CD16+ monocytes. To test this, healthy donors (HDs) or SCD patient monocytes were treated with different doses of free heme or hemolysed RBCs. We found dose-dependent HO-1 induction (five-fold at 20mM heme) in purified CD16- monocytes, but surprisingly none in CD16+ subset. However, upon co-culture with human umbilical vein endothelial cells (HUVEC), continuous or prior exposure to heme induced HO-1hi expression exclusively in CD16+ monocytes (5 fold in HD and further two fold in SCD compared to non-heme treated cocultures, p Disclosures No relevant conflicts of interest to declare.

GATA-1 and PU.1 are essential hematopoietic transcription factors that control erythromegakaryocytic and myelolymphoid differentiation, respectively. These proteins antagonize each other through direct physical interaction to repress alternate lineage programs. We used immortalized Gata1− erythromegakaryocytic progenitor cells to study how PU.1/Sfpi1 expression is regulated by GATA-1 and GATA-2, a related factor that is normally expressed at earlier stages of hematopoiesis. Both GATA factors bind the PU.1/Sfpi1 gene at 2 highly conserved regions. In the absence of GATA-1, GATA-2 binding is associated with an undifferentiated state, intermediate level PU.1/Sfpi1 expression, and low-level expression of its downstream myeloid target genes. Restoration of GATA-1 function induces erythromegakaryocytic differentiation. Concomitantly, GATA-1 replaces GATA-2 at the PU.1/Sfpi1 locus and PU.1/Sfpi1 expression is extinguished. In contrast, when GATA-1 is not present, shRNA knockdown of GATA-2 increases PU.1/Sfpi1 expression by 3-fold and reprograms the cells to become macrophages. Our findings indicate that GATA factors act sequentially to regulate lineage determination during hematopoiesis, in part by exerting variable repressive effects at the PU.1/Sfpi1 locus.

Red blood cell (RBC) and platelet antigen determination by DNA methods (genotyping) has become an important part of the practice of transfusion medicine over the past decade.The results are reliable and highly correlated with serologic phenotyping results, and are superior in some situations. Application of RBC genotyping to provide more precise transfusion therapy for various patient populations will be discussed, with a particular emphasis on those requiring chronic RBC therapy or with complex serologic reactivity. Current DNA arrays can test for a large number of blood group antigens, but routine typing of the ABO and Rh systems is not currently feasible due to the large number of genetic mutations. Patients with sickle cell disease (SCD) are one of the most challenging populations to safely transfuse, in part due to high rates of alloimmunization against Rh antigens despite prophylactic matching for D, C, and E antigens. Genotyping of the RH loci has revealed that RHD and RHCE variants are prevalent in African Blacks. Nearly 90% of patients with SCD have at least one altered RH gene and contributes to Rh alloimmunization in patients with SCD. The role of RH variation and alloimmunization in SCD will be reviewed, and the feasibility and approach to genetic RBC matching of patients and donors will be discussed. The use of targeted DNA arrays, whole exome sequencing, or whole genome sequencing to identify common and variant RH alleles will be addressed, including limitations of each method. Disclosures No relevant conflicts of interest to declare.

Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. Disclosures No relevant conflicts of interest to declare.

Transcription factor GATA-1 is essential at multiple stages of hematopoiesis. Murine gene targeting and analysis of naturally occurring human mutations demonstrate that GATA-1 drives the maturation of committed erythroid precursors and megakaryocytes. Prior studies also suggest additional, poorly defined, roles for GATA-1 at earlier stages of erythromegakaryocytic differentiation. To investigate these functions further, we stimulated Gata1- murine embryonic stem-cell-derived hematopoietic cultures with thrombopoietin, a multistage cytokine. Initially, the cultures generated a wave of mutant megakaryocytes. However, these were rapidly overgrown by a unique population of thrombopoietin-dependent blasts that express immature markers and proliferate indefinitely. Importantly, on restoration of GATA-1 function, these cells differentiated into both erythroid and megakaryocytic lineages, suggesting that they represent bipotential progenitors. Identical cells are also present in vivo, as indicated by flow cytometry and culture analysis of fetal livers from Gata1- chimeric mice. Our findings indicate that loss of GATA-1 impairs the maturation of megakaryocyte-erythroid progenitors. This defines a new role for GATA-1 at a relatively early stage of hematopoiesis and provides potential insight into recent discoveries that human GATA1 mutations promote acute megakaryoblastic leukemia, a clonal malignancy with features of both erythroid and megakaryocyte maturation.