Rare antigen‐negative red blood cells from pluripotent stem cells for precision transfusion medicine

Transfusion · 2026-04-24

BACKGROUND: Blood bank identification of antibodies against high-prevalence antigens remains a challenge due to the scarcity of antigen-negative reagent red cells sourced from blood donors. The MAM antigen, encoded by EMP3, is one such antigen associated with red cell alloimmunization and hemolytic disease of the fetus and newborn. STUDY DESIGN AND METHODS: We used CRISPR-Cas9 gene editing to generate an EMP3 knockout (EMP3KO) induced pluripotent stem cell (iPSC) line from a type O, Rh null parent line, enabling production of rare MAM-negative red blood cells. Since a prior study suggested that loss of EMP3 may enhance erythroid proliferation, we hypothesized that EMP3KO could both yield a rare reagent cell and potentially improve erythroid expansion to support scalable production. Transcriptomic analysis allowed us to further investigate the effect of EMP3 loss in late erythroblasts. RESULTS: EMP3KO cells differentiated efficiently into erythroid cells, showing >95% CD235/CD71 co-expression and orthochromatic erythroblast morphology. Compared to unedited cells, no proliferative advantage was observed, contrasting with prior non-isogenic cell models. Agglutination assays confirmed complete loss of MAM antigen and demonstrated the diagnostic utility for identifying MAM antibodies. Transcriptomic profiling of EMP3KO erythroblasts revealed expression of key erythroid genes, as well as regulators of proliferation and heme metabolism, was comparable to the parent line. DISCUSSION: This study demonstrates that iPSC technology combined with gene editing can generate rare antigen-negative RBCs for immunohematology applications. Beyond MAM, this platform offers a strategy to create additional rare RBC phenotypes, advancing precision transfusion medicine and improving antibody identification against high-prevalence antigens.

Understanding how a highly prevalent <i>GRK5</i> polymorphism affects platelets and enhances thrombotic risk

Blood · 2026-01-20 · 4 citations

ABSTRACT: Inherited genetic variants that modulate platelet function contribute significantly to thrombotic disorders, yet their mechanisms and clinical implications remain underexplored. Two genome-wide association studies identified an A→G variant (rs10886430) in the first intron of G protein-coupled receptor kinase 5 (GRK5), found in homozygosity in ∼5 million Americans. The homozygous GRK5 GG genotype is associated with an increased risk of stroke and venous thromboembolism, but the mechanistic link between this variant and thrombotic risk has remained unclear. To investigate this, we identified 3 GG individuals. GRK5 protein levels in GG platelets were 90% lower than in AA controls. The significant reduction in GRK5 levels in GG platelets led to elevated platelet responsiveness to thrombin and a protease-activated receptor 1 (PAR1) agonist but not a PAR4 agonist. These findings were corroborated in GRK5-/- induced pluripotent stem cell-derived megakaryocytes, transgenic Grk5-deficient murine platelets, and AA platelets exposed to a GRK5 inhibitor. We demonstrated that PAR1 internalization was reduced in GG platelets, leading to enhanced PAR1 signaling. Under venous shear in an endothelialized microfluidic system, GG platelets exhibited increased accumulation, which was reversed by PAR1 inhibition with vorapaxar. In an arterial murine thrombosis model following human platelet infusion, GG platelets also showed enhanced thrombus formation in vivo. This study provides, to our knowledge, the first experimental evidence directly linking a highly prevalent human GRK5 variant to defective PAR1 regulation and increased thrombotic risk. Together, these findings establish that the GRK5 GG genotype confers increased thrombotic potential through impaired PAR1 desensitization, providing mechanistic insight that connects human genetics, thrombin receptor signaling, and thrombotic disease.

CRISPR-mediated transcriptional activation as a mutation-independent therapeutic strategy for <i>SYNGAP1</i> -related intellectual disability

bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-29 · 2 citations

Abstract Synaptic Ras GTPase-activating protein (SynGAP) regulates synaptic strength and neuronal signaling, with essential roles in cortical development and synaptic plasticity. Heterozygous loss-of-function variants in SYNGAP1 cause SYNGAP1 -related intellectual disability (SRID), a severe neurodevelopmental disorder characterized by epilepsy, developmental delay, and autism. SYNGAP1 mutations often result in haploinsufficiency, providing a strong rationale for gene-targeted therapies. However, no treatment currently addresses the underlying genetic cause of SRID. Here, we developed a CRISPR-mediated transcriptional activation (CRISPRa) approach to upregulate the functional Syngap1 allele in a SRID mouse model. CRISPRa activated Syngap1 , normalized SynGAP protein expression and downstream signaling, and rescued working memory deficits. We validated the translational potential of this strategy in human induced pluripotent stem cell (hiPSC)-derived excitatory cortical neurons. CRISPRa rescued SYNGAP1 in two distinct loss-of-function variant lines. Together, these findings demonstrate the feasibility of mutation-independent transcriptional activation as a therapeutic approach for SRID and its broader applicability to haploinsufficiency disorders.

Generation of CHOPi014-A from healthy adult peripheral blood mononuclear cells

Stem Cell Research · 2025-08-24

CHOPWT15is a control male induced pluripotent stem cell (iPSC) line that can be used to model genetic variants by creating an allelic series of isogenic lines using genome editing technologies (Hockemeyer and Jaenisch, 2016; Maguire et al., 2022; Pavani et al., 2022). An allelic series of iPSC lines provides the means to perform genotype and phenotype analyses of pathological variants without the confounding effects of genetic background. Peripheral blood mononuclear cells (PBMCs), obtained from a healthy adult male, were reprogrammed using Sendai virus creating an iPSC line that has undetectable copy number variations (CNVs, resolution >= 100 kb) and differentiates to all three germlayers.

Single-cell transcriptomics reveal individual and cooperative effects of trisomy 21 and GATA1s on hematopoiesis

Stem Cell Reports · 2025-07-17 · 1 citations

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.

Using iPSC-derived hematopoietic stem cells with long-term engraftment capability to model fetal blood disorders

Blood · 2025-11-03

Abstract Introduction: Human developmental hematopoiesis is a complex process occurring in sequential waves at different embryonic sites. This process yields both differentiated blood cells essential for embryonic development and hematopoietic stem cells (HSCs) necessary for lifelong blood cell production. There are at least two main hematopoietic programs during embryogenesis: a transient primitive wave that primarily generates myelo-erythroid progenitors in the yolk sac, and a definitive wave occurring in the aorta-gonad-mesonephros region which produces progenitors with expanded lineage potential and the first transplantable HSCs. A challenge in generating HSCs from induced pluripotent stem cells (iPSCs) is that many current methods recapitulate early developmental stages only, failing to produce transplantable HSCs and limiting their application for in vivo studies. We have previously shown that we can recapitulate fetal erythropoiesis using a protocol that produces definitive hematopoietic cells. Red cells generated with this method primarily produce fetal globin and are functionally distinct from primitive erythroblasts. Here, we adapted this method to a serum-free 3D culture system to produce definitive hematopoietic stem progenitor cells (HSPCs) from iPSCs to 1) evaluate long-term engraftment via xenotransplantation and 2) model a preleukemic disorder of fetal origin, transient abnormal myelopoiesis (TAM), which affects ~20% of neonates with Trisomy 21 (T21) and is associated with mutations in the key hematopoietic transcription factor GATA1. Methods We designed a serum-free 3D culture system that directs mesodermal commitment through Wnt pathway activation by manipulating developmental cues through retinoic acid signaling and shear stress. We transplanted 24 NBSGW mice with 1.2-2 million iPSC-derived HSPCs using 3 distinct wildtype (WT) lines and experimental batches. The 3D definitive differentiation was assessed using isogenic T21 iPSCs with wild type GATA1 or the truncated isoform lacking the N-terminus, GATA1s. Results Long-term repopulation was achieved up to 20 weeks post-transplant in the bone marrow (BM) of all engrafted mice (mean = 3.9±11.7% human HLA-ABC+) and in the peripheral blood of 16/24 mice. In mice with the highest level of engraftment (&amp;gt;10%), we detected human HSCs as well as myeloid, lymphoid, erythroid and megakaryocyte precursors in the BM, indicating complete hematopoietic reconstitution. Human CD34+ cells recovered from the graft retained multilineage potential in colony forming assays. Importantly, no leukemia or teratomas were observed in any of the transplanted mice. To assess whether this definitive iPSC culture protocol could faithfully model the fetal blood disorder TAM, we generated definitive HSPCs from T21/wtGATA1 and T21/GATA1s iPSCs. Both T21 lines generated budding HSPCs at day 15; however, the percentage of CD34+CD45+ HSPCs was lower compared to euploid controls (60.5% of WT ±12.9 for T21, and 64.5% of WT ± 0.06 for T21/GATA1s). In addition, at day 15 we observed a significant CD41+CD42b+ megakaryocyte population in both trisomy lines, consistent with the disease phenotype and that was not previously observed with our primitive hematopoietic differentiation protocols. Compared to WT controls, we observed a 2.3-fold and a 4.2-fold increased megakaryocyte population from T21/wtGATA1 and T21/GATA1s iPSCs, respectively. T21/GATA1s HPSCs also showed aberrant morphology with megakaryocytic and blast-like features. Conclusions These studies show that our serum-free 3D culture system can generate iPSC-derived HSPCs that can engraft immunodeficient mice, reconstitute different blood lineages, and persist for 20 weeks. Using a T21/TAM iPSC model, we found an enhanced megakaryocyte population in T21/wtGATA1 consistent with a phenotype we observed with primary human fetal liver hematopoiesis, but not with iPSC-derived primitive hematopoietic progenitors. Thus, T21/GATA1s definitive HSPCs recapitulate the enhanced megakaryopoiesis consistent with TAM in vitro. Ongoing xenotransplant experiments will help elucidate the interaction of trisomy 21 and GATA1s and provide a novel in vivo model to study and treat human hematological diseases.

Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture

Stem Cell Reports · 2024-08-23 · 21 citations

While guided human cortical organoid (hCO) protocols reproducibly generate cortical cell types at one site, variability in hCO phenotypes across sites using a harmonized protocol has not yet been evaluated. To determine the cross-site reproducibility of hCO differentiation, three independent research groups assayed hCOs in multiple differentiation replicates from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol across 3 months. hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and expression of metabolism and cellular stress genes. Variability in hCO phenotypes correlated with stem cell gene expression prior to differentiation and technical factors associated with seeding, suggesting iPSC quality and treatment are important for differentiation outcomes. Cross-site reproducibility of hCO cell type proportions and organization encourages future prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

Rigor and reproducibility in human brain organoid research: Where we are and where we need to go

Stem Cell Reports · 2024-05-16 · 55 citations

Human brain organoid models have emerged as a promising tool for studying human brain development and function. These models preserve human genetics and recapitulate some aspects of human brain development, while facilitating manipulation in an in vitro setting. Despite their potential to transform biology and medicine, concerns persist about their fidelity. To fully harness their potential, it is imperative to establish reliable analytic methods, ensuring rigor and reproducibility. Here, we review current analytical platforms used to characterize human forebrain cortical organoids, highlight challenges, and propose recommendations for future studies to achieve greater precision and uniformity across laboratories.

3172 – SINGLE-CELL TRANSCRIPTOMICS REVEAL PERTURBATIONS IN HEMATOPOIETIC LINEAGE MATURATION DRIVEN BY TRISOMY 21 AND GATA1S

Experimental Hematology · 2024-08-01

Generation of a fluorescent mNeonGreen insulin reporter line in the H1 (WA01) hESC background

Stem Cell Research · 2024-09-11 · 1 citations

Over the past decade, the use of human stem cell-derived β cells (SC-β cells) to model pancreatic β cell development, function and disease has become increasingly common. Though protocols are rapidly improving, current directed differentiation strategies do not yield a pure population of insulin-positive SC-β cells in vitro. Therefore, it is experimentally advantageous to have reporter lines that allow for live sorting of insulin-positive populations. To aid in these studies, we have knocked mNeonGreen fluorescent protein into the endogenous insulin locus of the commonly used H1 (WA01) human embryonic stem cell line.