Foxa2 and H2A.Z mediate nucleosome depletion during embryonic stem cell differentiation.

Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions.

The use of pluripotent stem cells to generate diagnostic tools for transfusion medicine.

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.

Retinoic acid signaling within pancreatic endocrine progenitors regulates mouse and human ß cell specification.

Retinoic acid (RA) signaling is essential for multiple developmental processes, including appropriate pancreas formation from the foregut endoderm. RA is also required to generate pancreatic progenitors from human pluripotent stem cells. However, the role of RA signaling during endocrine specification has not been fully explored. In this study, we demonstrate that the disruption of RA signaling within the NEUROG3-expressing endocrine progenitor population impairs mouse ß cell differentiation and induces ectopic expression of crucial δ cell genes, including somatostatin. In addition, the inhibition of the RA pathway in hESC-derived pancreatic progenitors downstream of NEUROG3 induction impairs insulin expression. We further determine that RA-mediated regulation of endocrine cell differentiation occurs through Wnt pathway components. Together, these data demonstrate the importance of RA signaling in endocrine specification and identify conserved mechanisms by which RA signaling directs pancreatic endocrine cell fate.

RUNX-1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells.

Patients with familial platelet disorder with a predisposition to myeloid malignancy (FPDMM) harbor germline monoallelic mutations in a key hematopoietic transcription factor, RUNX-1. Previous studies of FPDMM have focused on megakaryocyte (Mk) differentiation and platelet production and signaling. However, the effects of RUNX-1 haploinsufficiency on hematopoietic progenitor cells (HPCs) and subsequent megakaryopoiesis remains incomplete. We studied induced pluripotent stem cell (iPSC)-derived HPCs (iHPCs) and Mks (iMks) from both patient-derived lines and a wild-type (WT) line modified to be RUNX-1 haploinsufficient (RUNX-1+/-), each compared with their isogenic WT control. All RUNX-1+/- lines showed decreased iMk yield and depletion of an Mk-biased iHPC subpopulation. To investigate global and local gene expression changes underlying this iHPC shift, single-cell RNA sequencing was performed on sorted FPDMM and control iHPCs. We defined several cell subpopulations in the Mk-biased iHPCs. Analyses of gene sets upregulated in FPDMM iHPCs indicated enrichment for response to stress, regulation of signal transduction, and immune signaling-related gene sets. Immunoblot analyses in FPDMM iMks were consistent with these findings, but also identified augmented baseline c-Jun N-terminal kinase (JNK) phosphorylation, known to be activated by transforming growth factor-ß1 (TGF-ß1) and cellular stressors. These findings were confirmed in adult human CD34+-derived stem and progenitor cells (HSPCs) transduced with lentiviral RUNX1 short hairpin RNA to mimic RUNX-1+/-. In both iHPCs and CD34+-derived HSPCs, targeted inhibitors of JNK and TGF-ß1 pathways corrected the megakaryopoietic defect. We propose that such intervention may correct the thrombocytopenia in patients with FPDMM.

Modeling genetic platelet disorders with human pluripotent stem cells: mega-progress but wanting more on our plate(let).

PURPOSE OF REVIEW: Megakaryocytes are rare hematopoietic cells that play an instrumental role in hemostasis, and other important biological processes such as immunity and wound healing. With the advent of cell reprogramming technologies and advances in differentiation protocols, it is now possible to obtain megakaryocytes from any pluripotent stem cell (PSC) via hematopoietic induction. Here, we review recent advances in PSC-derived megakaryocyte (iMK) technology, focusing on platform validation, disease modeling and current limitations. RECENT FINDINGS: A comprehensive study confirmed that iMK can recapitulate many transcriptional and functional aspects of megakaryocyte and platelet biology, including variables associated with complex genetic traits such as sex and race. These findings were corroborated by several pathological models in which iMKs revealed molecular mechanisms behind inherited platelet disorders and assessed the efficacy of novel pharmacological interventions. However, current differentiation protocols generate primarily embryonic iMK, limiting the clinical and translational potential of this system. SUMMARY: iMK are strong candidates to model pathologic mutations involved in platelet defects and develop innovative therapeutic strategies. Future efforts on generating definitive hematopoietic progenitors would improve current platelet generation protocols and expand our capacity to model neonatal and adult megakaryocyte disorders.

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis.

AIMS/HYPOTHESIS: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. METHODS: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. RESULTS: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. CONCLUSIONS/INTERPRETATION: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. DATA AVAILABILITY: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.

Tropomyosin 1 genetically constrains in vitro hematopoiesis.

BACKGROUND: Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits. RESULTS: Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields. CONCLUSIONS: Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.

Identifying and enriching platelet-producing human stem cell-derived megakaryocytes using factor V uptake.

Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.

Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification.

Endoderm cells undergo sequential fate choices to generate insulin-secreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells.

Understanding platelet generation from megakaryocytes: implications for in vitro-derived platelets.

Platelets are anucleate cytoplasmic discs derived from megakaryocytes that circulate in the blood and have major roles in hemostasis, thrombosis, inflammation, and vascular biology. Platelet transfusions are required to prevent the potentially life-threatening complications of severe thrombocytopenia seen in a variety of medical settings including cancer therapy, trauma, and sepsis. Platelets used in the clinic are currently donor-derived which is associated with concerns over sufficient availability, quality, and complications due to immunologic and/or infectious issues. To overcome our dependence on donor-derived platelets for transfusion, efforts have been made to generate in vitro-based platelets. Work in this area has advanced our understanding of the complex processes that megakaryocytes must undergo to generate platelets both in vivo and in vitro. This knowledge has also defined the challenges that must be overcome to bring in vitro-based platelet manufacturing to a clinical reality. This review will focus on our understanding of committed megakaryocytes and platelet release in vivo and in vitro, and how this knowledge can guide the development of in vitro-derived platelets for clinical application.

Induced Pluripotent Stem Cell-Derived Megakaryocytes and Platelets for Disease Modeling and Future Clinical Applications.

Platelets, derived from megakaryocytes, are anucleate cytoplasmic discs that circulate in the blood stream and play major roles in hemostasis, inflammation, and vascular biology. Platelet transfusions are used in a variety of medical settings to prevent life-threatening thrombocytopenia because of cancer therapy, other causes of acquired or inherited thrombocytopenia, and trauma. Currently, platelets used for transfusion purposes are donor derived. However, there is a drive to generate nondonor sources of platelets to help supplement donor-derived platelets. Efforts have been made by many laboratories to generate in vitro platelets and optimize their production and quality. In vitro-derived platelets have the potential to be a safer, more uniform product, and genetic manipulation could allow for better treatment of patients who become refractory to donor-derived units. This review focuses on potential clinical applications of in vitro-derived megakaryocytes and platelets, current methods to generate and expand megakaryocytes from pluripotent stem cell sources, and the use of these cells for disease modeling.

Comparative analysis of human ex vivo-generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale.

Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application.

Transcriptional competence and the active marking of tissue-specific enhancers by defined transcription factors in embryonic and induced pluripotent stem cells.

We reported previously that well-characterized enhancers but not promoters for typical tissue-specific genes, including the classic Alb1 gene, contain unmethylated CpG dinucleotides and evidence of pioneer factor interactions in embryonic stem (ES) cells. These properties, which are distinct from the bivalent histone modification domains that characterize the promoters of genes involved in developmental decisions, raise the possibility that genes expressed only in differentiated cells may need to be marked at the pluripotent stage. Here, we demonstrate that the forkhead family member FoxD3 is essential for the unmethylated mark observed at the Alb1 enhancer in ES cells, with FoxA1 replacing FoxD3 following differentiation into endoderm. Up-regulation of FoxD3 and loss of CpG methylation at the Alb1 enhancer accompanied the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Studies of two genes expressed in specific hematopoietic lineages revealed that the establishment of enhancer marks in ES cells and iPS cells can be regulated both positively and negatively. Furthermore, the absence of a pre-established mark consistently resulted in resistance to transcriptional activation in the repressive chromatin environment that characterizes differentiated cells. These results support the hypothesis that pluripotency and successful reprogramming may be critically dependent on the marking of enhancers for many or all tissue-specific genes.

High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia.

Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin αIIbß3. Hemizygous insertion of Gp1ba promoter-driven human αIIb complementary DNA into the AAVS1 locus of iPSCs led to high αIIb messenger RNA and protein expression and correction of surface αIIbß3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin αIIbß3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function.

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients.

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.

The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation.

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.

Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell lines.

Induced pluripotent stem cells (iPSCs) hold great promise for modeling human hematopoietic diseases. However, intrinsic variability in the capacities of different iPSC lines for hematopoietic development complicates comparative studies and is currently unexplained. We created and analyzed 3 separate iPSC clones from fibroblasts of 3 different normal individuals using a standardized approach that included excision of integrated reprogramming genes by Cre-Lox mediated recombination. Gene expression profiling and hematopoietic differentiation assays showed that independent lines from the same individual were generally more similar to one another than those from different individuals. However, one iPSC line (WT2.1) exhibited a distinctly different gene expression, proliferation rate, and hematopoietic developmental potential relative to all other iPSC lines. This "outlier" clone also acquired extensive copy number variations (CNVs) during reprogramming, which may be responsible for its divergent properties. Our data indicate how inherent and acquired genetic differences can influence iPSC properties, including hematopoietic potential.

MicroRNA screen of human embryonic stem cell differentiation reveals miR-105 as an enhancer of megakaryopoiesis from adult CD34+ cells.

MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96-well plate electroporation, we screened 466 human miRNA mimics by four-color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4-5 days post-transfection, flow cytometry of erythroid (CD235(+)CD41(-)), megakaryocyte (CD41(+)CD42(+)), and myeloid (CD18(+)CD235(-)) lineages revealed miR-105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC-derived CMPs, miR-105 caused a sixfold enhancement in megakaryocyte production. miR-513a, miR-571, and miR-195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR-105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34(+) cells derived from peripheral blood. In addition, adult CD34(+) cells express endogenous miR-105 during megakaryocyte differentiation. siRNA knockdown of the hematopoietic transcription factor c-Myb caused a similar enhancement of megakaryocyte production as miR-105. Finally, a luciferase/c-Myb-3'UTR construct and Western blot analysis demonstrated that the hematopoietic transcription factor c-Myb mRNA was a target of miR-105. We report a novel hESC-based miR screening platform and demonstrate that miR-105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis.

Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cells.

Patients with Down syndrome (trisomy 21, T21) have hematologic abnormalities throughout life. Newborns frequently exhibit abnormal blood counts and a clonal preleukemia. Human T21 fetal livers contain expanded erythro-megakaryocytic precursors with enhanced proliferative capacity. The impact of T21 on the earliest stages of embryonic hematopoiesis is unknown and nearly impossible to examine in human subjects. We modeled T21 yolk sac hematopoiesis using human induced pluripotent stem cells (iPSCs). Blood progenitor populations generated from T21 iPSCs were present at normal frequency and proliferated normally. However, their developmental potential was altered with enhanced erythropoiesis and reduced myelopoiesis, but normal megakaryocyte production. These abnormalities overlap with those of T21 fetal livers, but also reflect important differences. Our studies show that T21 confers distinct developmental stage- and species-specific hematopoietic defects. More generally, we illustrate how iPSCs can provide insight into early stages of normal and pathological human development.

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.