Primitive Hematopoiesis Research Articles

Comparative analysis of iPSC-derived NK cells from two differentiation strategies reveals distinct signatures and cytotoxic activities.

The ability to generate natural killer (NK) cells from induced pluripotent stem cells (iPSCs) has given rise to new possibilities for the large-scale production of homogeneous immunotherapeutic cellular products and opened new avenues towards the creation of "off-the-shelf" cancer immunotherapies. However, the differentiation of NK cells from iPSCs remains poorly understood, particularly regarding the ontogenic landscape of iPSC-derived NK (iNK) cells produced in vitro and the influence that the differentiation strategy employed may have on the iNK profile. To investigate this question, we conducted a comparative analysis of two sets of iNK cells generated from the same iPSC line using two different protocols: (i) a short-term, clinically compatible feeder-free protocol corresponding to primitive hematopoiesis, and (ii) a lymphoid-based protocol representing the definitive hematopoietic step. Our work demonstrated that both protocols are capable of producing functional iNK cells. However, the two sets of resulting iNKs exhibited distinct phenotypes and transcriptomic profiles. The lymphoid-based differentiation approach generated iNKs with a more mature and activated profile, which demonstrated higher cytotoxicity against cancer cell lines compared to iNK cells produced under short-term feeder-free conditions suggesting that the differentiation strategy must be considered when designing iNK cell-based adoptive immunotherapies.

Blood-generating heart-forming organoids recapitulate co-development of the human haematopoietic system and the embryonic heart.

Despite the biomedical importance of haematopoietic stem cells and haematopoietic progenitor cells, their in vitro stabilization in a developmental context has not been achieved due to limited knowledge of signals and markers specifying the multiple haematopoietic waves as well as ethically restricted access to the human embryo. Thus, an in vitro approach resembling aspects of haematopoietic development in the context of neighbouring tissues is of interest. Our established human pluripotent stem cell-derived heart-forming organoids (HFOs) recapitulate aspects of heart, vasculature and foregut co-development. Modulating HFO differentiation, we here report the generation of blood-generating HFOs. While maintaining a functional ventricular-like heart anlagen, blood-generating HFOs comprise a mesenchyme-embedded haemogenic endothelial layer encompassing multiple haematopoietic derivatives and haematopoietic progenitor cells with erythro-myeloid and lymphoid potential, reflecting aspects of primitive and definitive haematopoiesis. The model enables the morphologically structured co-development of cardiac, endothelial and multipotent haematopoietic tissues equivalent to the intra-embryonic haematopoietic region in vivo, promoting research on haematopoiesis in vitro.

Hypoxia-sonic hedgehog axis as a driver of primitive hematopoiesis development and evolution in cavefish

The teleost Astyanax mexicanus consists of surface dwelling (surface fish) and cave dwelling (cavefish) forms. Cavefish have evolved in subterranean habitats characterized by reduced oxygen levels (hypoxia) and exhibit a subset of phenotypic traits controlled by increased Sonic hedgehog (Shh) signaling along the embryonic midline. The enhancement of primitive hematopoietic domains, which are formed bilaterally in the anterior and posterior lateral plate mesoderm, are responsible for the development of more larval erythrocytes in cavefish relative to surface fish. In this study, we determine the role of hypoxia and Shh signaling in the development and evolution of primitive hematopoiesis in cavefish. We show that hypoxia treatment during embryogenesis increases primitive hematopoiesis and erythrocyte development in surface fish. We also demonstrate that upregulation of Shh midline signaling by the Smoothened agonist SAG increases primitive hematopoiesis and erythrocyte development in surface fish, whereas Shh downregulation via treatment with the Smoothened inhibitor cyclopamine decreases these traits in cavefish. Together these results suggest that hematopoietic enhancement is regulated by hypoxia and Shh signaling. Lastly, we demonstrate that hypoxia enhances expression of Shh signaling along the midline of surface fish embryos. We conclude that hypoxia-mediated Shh plasticity may be a driving force for the adaptive evolution of primitive hematopoiesis and erythrocyte development in cavefish.

Hypoxia-Sonic Hedgehog Axis as a Driver of Primitive Hematopoiesis Development and Evolution in Cavefish.

Hypoxia increases hematopoiesis and erythrocytes in surface fishShh upregulation increases hematopoiesis and erythrocytes in surface fishShh inhibition decreases hematopoiesis and erythrocytes in cavefishHypoxia upregulates Shh along the embryonic midline in surface fish.

Zebrafish nampt-a mutants are viable despite perturbed primitive hematopoiesis.

Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD+ in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been shown to cause abnormal development and defective hematopoiesis concomitant with decreased NAD+ levels. However, surprisingly, nampt-a mutant zebrafish were recently found to be viable, suggesting a discrepancy between the phenotypes in knockdown and knockout conditions. Here, we address this discrepancy by directly comparing loss-of-function approaches that result in identical defective transcripts in morphants and mutants. Using CRISPR/Cas9-mediated mutagenesis, we generated nampt-a mutant lines that carry the same mis-spliced mRNA as nampt-a morphants. Despite reduced NAD+ levels and perturbed expression of specific blood markers, nampt-a mutants did not display obvious developmental defects and were found to be viable. In contrast, injection of nampt-a morpholinos into wild-type or mutant nampt-a embryos caused aberrant phenotypes. Moreover, nampt-a morpholinos caused additional reduction of blood-related markers in nampt-a mutants, suggesting that the defects observed in nampt-a morphants can be partially attributed to off-target effects of the morpholinos. Our findings show that zebrafish nampt-a mutants are viable despite reduced NAD+ levels and a perturbed hematopoietic gene expression program, indicating strong robustness of primitive hematopoiesis during early embryogenesis.

Stat3 Regulates Developmental Hematopoiesis and Impacts Myeloid Cell Function via Canonical and Non-Canonical Modalities

Introduction: Signal transducer and activator of transcription (STAT) 3 is extensively involved in the development, homeostasis, and function of immune cells, with STAT3 disruption associated with human immune-related disorders. The roles ascribed to STAT3 have been assumed to be due to its canonical mode of action as an inducible transcription factor downstream of multiple cytokines, although alternative noncanonical functional modalities have also been identified. The relative involvement of each mode was further explored in relevant zebrafish models. Methods: Genome editing with CRISPR/Cas9 was used to generate mutants of the conserved zebrafish Stat3 protein: a loss of function knockout (KO) mutant and a mutant lacking C-terminal sequences including the transactivation domain (ΔTAD). Lines harboring these mutations were analyzed with respect to blood and immune cell development and function in comparison to wild-type zebrafish. Results: The Stat3 KO mutant showed perturbation of hematopoietic lineages throughout primitive and early definitive hematopoiesis. Neutrophil numbers did not increase in response to lipopolysaccharide (LPS) or granulocyte colony-stimulating factor (G-CSF) and their migration was significantly diminished, the latter correlating with abrogation of the Cxcl8b/Cxcr2 pathway, with macrophage responses perturbed. Intriguingly, many of these phenotypes were not shared by the Stat3 ΔTAD mutant. Indeed, only neutrophil and macrophage development were disrupted in these mutants with responsiveness to LPS and G-CSF maintained, and neutrophil migration actually increased. Conclusion: This study has identified roles for zebrafish Stat3 within hematopoietic stem cells impacting multiple lineages throughout primitive and early definitive hematopoiesis, myeloid cell responses to G-CSF and LPS and neutrophil migration. Many of these roles showed conservation, but notably several involved noncanonical modalities, providing additional insights for relevant diseases.

Ebf1 Is Involved in Primitive Myelopoiesis and Contributes in Leukemia Pathogenesis

Acute myeloid leukemia (AML) is caused by the abnormal proliferation and differentiation of a clonal population of myeloid progenitors. EBF1 is a key transcriptional factor in regulating hematopoiesis and has been linked to acute lymphoblastic leukemia (ALL). However, the relationship between AML and EBF1 mutation has not yet been studied. Recent research has shown that deleting EBF1 results in myeloid-biased progenitors and decreased HSC quiescence and repopulation capacity. This suggests that EBF1 is involved in earlier hematopoiesis and has additional functions beyond B-cell committed development. Our clinical center conducted whole-exome sequencing on 231 cases of acute myeloid leukemia and identified four patients with an Ebf1 mutation. This corresponds to a mutation frequency of 1.73% and the mutations were identified as p.Q477H (c.1434G>T), p.S513P (c.1537T>C), p.C534F (c.1601GT), and p.P570A (c.1708C>G). Data from the cBioPortal database showed that AML patients with Ebf1 deep deleted mutation had shortened survival. To better understand the role of Ebf1 in AML pathogenesis, we generated an ebf1a-deficient zebrafish mutant. Further research showed that the ebf1a deficiency caused neutrophilia during primitive hematopoiesis. BrdU staining in Tg(coronin1a: GFP) labeled myeloid progenitors was significantly higher in ebf1a-deficient larvae than in wild-type larvae, indicating that ebf1a deficiency led to increased proliferating myeloid progenitors which would take responsibility for the neutrophilia in early-developing larvae. We conducted experiments using four RNA variants (p.Q477H, p.S513P, p.C534F, and p.P570A) injected into ebf1a deficiency zebrafish larvae to verify their pathogenicity. Our findings showed that while EBF1 p.Q477H and p.S513P caused a decrease in neutrophils, p.C534F resulted in neutrophilia, indicating that it might be a loss-of-function mutation. To further understand the mechanism, we introduced EBF1 p.C534F to AML cell culture, and CCk8 and cell cycle analysis showed increased proliferation rates and disturbed cell cycle balance, leading to increased proliferation of AML cells of EBF1-C534F mutation. The cells bearing this mutation showed a higher rate of proliferation, likely due to a disturbance in the balance of the cell cycle. These findings provide valuable insight into the potential pathogenetic mechanism of EBF1 p.C534F. Our investigation into the cause of ebf1 deficiency disrupting normal hematopoiesis led us to perform bulk RNA-sequencing with sorted myeloid-progenitors labeled by Tg:(coronin1a-GFP) in 30hpf zebrafish larvae. We found that leukocyte differentiation and myeloid cell cycle-related genes such as lyz, lygl1, coro1a, lcp1, cebpa, spi1b, mpx, irf8, il6, il34, etc were upregulated in ebf1a deficiency zebrafish. Additionally, anxa1c, anxa1b, Cish, and nlrc3l, which have been reported to suppress lymphoid proliferation but increase myeloid expansion, were down-regulated. Go enrichment analysis showed that the significant genes were associated with hematopoietic disorders including AML and MDS. To address the issue of disturbed cell cycle balance caused by EBF1 deficiency and loss-of-function mutation, we sought to suppress the G1-S transition via CDK inhibitor. We treated EBF1-C534F, EBF1-WT, and control AML cell culture with CDK4 inhibitor: AMG925. We were surprised to find that the CDK4 inhibitor sufficiently suppressed the proliferation and reversed the prolonged S phase caused by EBF1 mutation. We investigated the myeloid-bias function of Ebf1 deficiency and the potential pathogenesis of EBF1 mutations of AML from the development view and were surprised to find CDK inhibitors as a potential drug for EBF1-deficient AML patients.

Stag2a or Stag2b Knockout Zebrafish Are Viable, Yet Loss of Stag2a Is Associated with Abnormal Expression of Early and Late Myeloid and Lymphoid Markers

Introduction:Mutations in Stromal antigen 2 ( STAG2) are frequently found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In mice, loss of Stag2 is embryonic lethal. Conditional knockout of Stag2 in adult mice results in MDS-like hematopoietic anomalies. Zebrafish harbor two paralogues of STAG2 ( stag2a and stag2b). To date, a complete stag2-null zebrafish model organism has not been developed. Recently reported, a stag2b-/- zebrafish demonstrated downregulation in primitive erythropoiesis. However, any hematopoiesis beyond 2 days post fertilization (2 dpf) was not detailed. Furthermore, a stag2a mutant zebrafish has not been reported. Methods and Results: Using CRISPR/Cas9 genome editing, we created a stag2a mutant anda stag2b mutant zebrafish. Both mutations result in a frame shift and premature protein truncations. Unlike the murine Stag2 -/- mutant, zebrafish stag2a -/- mutants and stag2b -/-mutants survive to adulthood, with no obvious morphological phenotypes. Detection of hemoglobin, using O-dianisidine staining, reveals anemia at 31 hours post fertilization (31 hpf) in stag2a +/- mutants (10/29), stag2a -/-mutants (10/15), stag2b +/-mutants (6/11), and stag2b -/-mutants (5/7); wildtype siblings appear unaffected. Intriguingly, at 4 dpf, stag2a maternal-zygotic mutants show increased expression levels of early and late myeloid and lymphoid markers. ( Figure 1) Work is ongoing to analyze blood cell lineages from zebrafish whole kidney marrow, the equivalent of mammalian bone marrow, in adult stag2a and adult stag2b mutants. In addition, we are characterizing the zebrafish stag2a;stag2b double mutants, which we will present. Conclusions: In zebrafish, stag2a and stag2b contribute to proper primitive and definitive hematopoiesis. Our organismal model of Stag2 deletion in the zebrafish will yield new insight into how loss of STAG2 may lead to MDS/AML.

Sclerotome-derived PDGF signaling functions as a niche cue responsible for primitive erythropoiesis.

Primitive erythropoiesis serves a vital role in embryonic development, generating primitive red blood cells responsible for transportation of oxygen throughout the body. Although diverse niche factors are known to function in definitive hematopoiesis, the microenvironment contributing to primitive hematopoiesis remains largely elusive. Here, we report that platelet-derived growth factor (PDGF) signaling is required for erythroid progenitor differentiation in zebrafish. Ablating pdgfαa (also known as pdgfaa) and pdgfαb (also known as pdgfab) or blocking PDGF signaling with an inhibitor impairs erythroid progenitor differentiation, thus resulting in a significant decrease in the number of erythrocytes. We reveal that pdgfαb is expressed in sclerotomal cells, and that its receptor genes, pdgfra and pdgfrb, are expressed in the adjacent erythroid progenitor cells. Sclerotome-specific overexpression of pdgfαb effectively restores primitive erythropoiesis in pdgfαa-/-;pdgfαb-/- mutant embryos. In addition, we have defined ERK1/2 signaling as a downstream pathway of PDGF signaling during embryonic erythropoiesis. Taken together, our findings indicate that PDGF signaling derived from sclerotome functions as a niche cue for primitive erythropoiesis.

Tropomyosin 1 Deficiency Facilitates Epithelial-to-Mesenchymal Transitions to Promote Hemogenic Endothelial Specification and Hematopoietic Progenitor Cell Formation

Tropomyosins coat actin filaments and impact many actin-related biology, including signaling and cell morphogenesis. 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, single cell transcriptomics, and a novel murine model, we aimed to define how and when TPM1 regulated developmental hematopoiesis. We used CRISPR/Cas9 to create TPM1 knockout (KO) iPSC lines in a primitive hematopoiesis system, in which hemogenic endothelial cells spawn hematopoietic progenitor cells (HPC). Western blot showed that TPM1 was highly expressed in iPSCs and endothelial cells but virtually absent in HPCs. TPM1KO enhanced endothelial cell and hematopoietic progenitor cell (HPC) formation without augmenting cell cycle kinetics. RNA sequencing analysis showed that TPM1KO altered epithelial-to-mesenchymal transition (EMT)-related gene expression and functionally enhanced EMT during in vitro hematopoiesis, without generally disrupting normal cell development. Single-cell transcriptomics showed that TPM1 was highly expressed in human and mouse hemogenic endothelium, and abrogated in HPCs, suggesting that TPM1 might also regulate developmental hematopoiesis in vivo similar to our in vitro findings. Indeed, TPM1 expression coincided with EMT-related gene expression during in vivo hematopoiesis (p<1x10-16). To investigate the role of TPM1 in vivo, we used a novel murine TPM1 GeneTrap-Reporter mouse model. By western blot, TPM1 protein was decreased ~50% in peripheral blood. TPM1 GT/GTmice displayed embryonic lethality by E9.5, due to cardiac dysmorphology previously seen in TPM1 knockout mice. TPM1 reporter expression (LacZ) was observed in E9.5 endothelium, temporally consistent with Runx1+/CD31+ hemogenic endothelial cell emergence. Whole-mount imaging of TPM1 GT/+ embryos showed an enhanced percentage (%) of Runx1 + HE at E9.5 and Runx1 +cKit + HPCs at E10.5. Consistent with increased embryonic HPC production, TPM1 GT/+mice showed increased percentages of Lin-Sca1+cKit+ HPCs in bone marrow at 6-8 weeks of age. Complete blood counts in 6-8 weeks-old mice were normal. Conclusions These findings illuminate novel effects of TPM1, a cytoskeletal regulatory molecule, on developmental hematopoiesis. TPM1KO disinhibits EMT to promote hemogenic endothelial specification and HPC production during developmental hematopoiesis, without compromising mature blood cell function. Temporal TPM1 inhibition may offer innovative approaches to enhance blood cell formation in translationally relevant cell culture systems. Our findings may explain genetic associations linking polymorphisms in the TPM1 gene locus with altered human blood traits. More broadly, the radical changes required to form HPCs from endothelial cells during hematopoiesis represent an exciting area of study for actin and tropomyosin biology.

Aggf1 Specifies Hemangioblasts at the Top of Regulatory Hierarchy via Npas4l and mTOR-S6K-Emp2-ERK Signaling.

Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins. Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown. These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.

Adducin 1 is essential for the survival of erythroid precursors via regulating p53 transcription in zebrafish

Adducin 1 (Add1) is known as a membrane cytoskeletal protein, but its nuclear function remains unclear. In this study, we generated add1-deficient zebrafish to investigate its role in hematopoiesis. Lack of add1 impaired both primitive and definitive hematopoiesis, preventing healthy erythrocyte development. RNA sequencing revealed activation of the p53 pathway in add1-depleted erythroblast cells, leading to apoptosis at the 14-somites stage and 24 hpf. Interestingly, partial rescue of the anemic phenotype and apoptosis was observed with p53 insufficiency. Mechanistically, ADD1 was found to regulate promoter activity. These findings demonstrate that Add1 plays a crucial role in zebrafish erythropoiesis, involving the p53-mediated apoptotic pathway, expanding its regulatory role beyond cytoskeletal functions.

IN VITRO PROPAGATION OF PLATELETS FROM EMBRYONIC STEM CELLS WITH C. PAPAYA LEAF EXTRACT

Objective: The depletion of platelets in patients under different clinical conditions like thrombocytopenia is often associated with a significant mortality factor. Alternative modes of platelet propagation from other resources, including stem cells, are evaluated under in vitro conditions. Embryonic stem (ES) cells can proliferate and differentiate into various cell types. This study evaluated the generation of functional platelets from embryonic stem cells with C. papaya leaf extract through a controlled sequential pathway to meet the clinical demand.
 Methods: In vitro culture of embryonic stem cells was carried out with the help of growth factors and a suitable medium. The differentiated megakaryocytes and platelets were analyzed by staining, flow cytometer and functional studies. The normal megakaryocytes were compared with the megakaryocytes differentiated from Embryonic Stem cells.
 Results: The platelets derived from embryonic stem cells are morphologically identical to normal platelets isolated from peripheral blood, and the number of viable cells was analyzed by flow cytometer. The distinctive two waves of platelet production represented normal primitive and definitive hematopoiesis.
 Conclusion: This study is a preliminary milestone in platelet propagation from embryonic stem cells with C. papaya leaf extract. The differentiation of ESCs into megakaryocyte-mediated platelets with C. papaya leaf extract has shown valuable information on the positive effects that embryonic stem cells can be used for large-scale platelet production.

LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis.

Transcription factor genetics and biology in predisposition to bone marrow failure and hematological malignancy.

Transcription factors (TFs) play a critical role as key mediators of a multitude of developmental pathways, with highly regulated and tightly organized networks crucial for determining both the timing and pattern of tissue development. TFs can act as master regulators of both primitive and definitive hematopoiesis, tightly controlling the behavior of hematopoietic stem and progenitor cells (HSPCs). These networks control the functional regulation of HSPCs including self-renewal, proliferation, and differentiation dynamics, which are essential to normal hematopoiesis. Defining the key players and dynamics of these hematopoietic transcriptional networks is essential to understanding both normal hematopoiesis and how genetic aberrations in TFs and their networks can predispose to hematopoietic disease including bone marrow failure (BMF) and hematological malignancy (HM). Despite their multifaceted and complex involvement in hematological development, advances in genetic screening along with elegant multi-omics and model system studies are shedding light on how hematopoietic TFs interact and network to achieve normal cell fates and their role in disease etiology. This review focuses on TFs which predispose to BMF and HM, identifies potential novel candidate predisposing TF genes, and examines putative biological mechanisms leading to these phenotypes. A better understanding of the genetics and molecular biology of hematopoietic TFs, as well as identifying novel genes and genetic variants predisposing to BMF and HM, will accelerate the development of preventative strategies, improve clinical management and counseling, and help define targeted treatments for these diseases.

Primitive haematopoiesis in the human placenta gives rise to macrophages with epigenetically silenced HLA-DR

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. Although this process is thought to be spatially restricted to the yolk sac in the mouse, in humans, it remains poorly understood. Human foetal placental macrophages, or Hofbauer cells (HBC), arise during the primitive haematopoietic wave ~18 days post conception and lack expression of human leukocyte antigen (HLA) class II. Here, we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro culture experiments we demonstrate that PEMP generate HBC-like cells lacking HLA-DR expression. We find the absence of HLA-DR in primitive macrophages is mediated via epigenetic silencing of class II transactivator, CIITA, the master regulator of HLA class II gene expression. These findings establish the human placenta as an additional site of primitive haematopoiesis.

The Role of SCL Isoforms in Embryonic Hematopoiesis

Three waves of hematopoiesis occur in the mouse embryo. The primitive hematopoiesis appears as blood islands in the extra embryonic yolk sac at E7.5. The extra embryonic pro-definitive hematopoiesis launches in late E8 and the embryonic definitive one turns on at E10.5 indicated by the emergence of hemogenic endothelial cells on the inner wall of the extra embryonic arteries and the embryonic aorta. To study the roles of SCL protein isoforms in murine hematopoiesis, the SCL-large (SCL-L) isoform was selectively destroyed with the remaining SCL-small (SCL-S) isoform intact. It was demonstrated that SCL-S was specifically expressed in the hemogenic endothelial cells (HECs) and SCL-L was only detected in the dispersed cells after budding from HECs. The SCLΔ/Δ homozygous mutant embryos only survived to E10.5 with normal extra embryonic vessels and red blood cells. In wild-type mouse embryos, a layer of neatly aligned CD34+ and CD43+ cells appeared on the endothelial wall of the aorta of the E10.5 fetus. However, the cells at the same site expressed CD31 rather than CD34 and/or CD43 in the E10.5 SCLΔ/Δ embryo, indicating that only the endothelial lineage was developed. These results reveal that the SCL-S is sufficient to sustain the primitive hematopoiesis and SCL-L is necessary to launch the definitive hematopoiesis.

Socs3b regulates the development and function of innate immune cells in zebrafish.

Suppressor of cytokine signaling 3 (SOCS3) is a critical component of the negative feedback regulation that controls signaling by cytokines and other factors thereby ensuring that important processes such as hematopoiesis and inflammation occur at appropriate levels. To gain further insights into SOCS3 function, the zebrafish socs3b gene was investigated through analysis of a knockout line generated using CRISPR/Cas9-mediated genome editing. Zebrafish socs3b knockout embryos displayed elevated numbers of neutrophils during primitive and definitive hematopoiesis but macrophage numbers were not altered. However, the absence of socs3b reduced neutrophil functionality but enhanced macrophage responses. Adult socs3b knockout zebrafish displayed reduced survival that correlated with an eye pathology involving extensive infiltration of neutrophils and macrophages along with immune cell dysregulation in other tissues. These findings identify a conserved role for Socs3b in the regulation of neutrophil production and macrophage activation.

Impact of the Aryl Hydrocarbon Receptor on Aurora A Kinase and the G2/M Phase Pathway in Hematopoietic Stem and Progenitor Cells

Recent evidence suggests that the environment-sensing transcription factor aryl hydrocarbon receptor (AHR) is an important regulator of hematopoiesis. Yet, the mechanisms and extent of AHR-mediated regulation within the most primitive hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), are poorly understood. Through a combination of transcriptomic and flow cytometric approaches, this study provides new insight into how the AHR influences hematopoietic stem and progenitor cells. Comparative analysis of intraphenotypic transcriptomes of hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells from AHR knockout (AHR KO) and wild type mice revealed significant differences in gene expression patterns. Notable among these were differences in expression of cell cycle regulators, specifically an enrichment of G2/M checkpoint genes when Ahr was absent. This included the regulator Aurora A kinase (Aurka, AurA). Analysis of AurA protein levels in HSPC subsets using flow cytometry, in combination with inducible AHR KO or in vivo AHR antagonism, showed that attenuation of AHR increased levels of AurA in HSCs and lineage-biased MPP cells. Overall, these data highlight a potential novel mechanism by which AHR controls HSC homeostasis and HSPC differentiation. These findings advance the understanding of how AHR influences and regulates primitive hematopoiesis.

Human myeloid progenitor glucocorticoid receptor activation causes genomic instability, type 1 IFN- response pathway activation and senescence in differentiated microglia; an early life stress model.

One form of early life stress, prenatal exposure to glucocorticoids (GCs), confers a higher risk of psychiatric and neurodevelopmental disorders in later life. Increasingly, the importance of microglia in these disorders is recognized. Studies on GCs exposure during microglial development have been limited, and there are few, if any, human studies. We established an in vitro model of ELS by continuous pre-exposure of human iPS-microglia to GCs during primitive hematopoiesis (the critical stage of iPS-microglial differentiation) and then examined how this exposure affected the microglial phenotype as they differentiated and matured to microglia, using RNA-seq analyses and functional assays. The iPS-microglia predominantly expressed glucocorticoid receptors over mineralocorticoid receptors, and in particular, the GR-α splice variant. Chronic GCs exposure during primitive hematopoiesis was able to recapitulate in vivo ELS effects. Thus, pre-exposure to prolonged GCs resulted in increased type I interferon signaling, the presence of Cyclic GMP-AMP synthase-positive (cGAS) micronuclei, cellular senescence and reduced proliferation in the matured iPS-microglia. The findings from this in vitro ELS model have ramifications for the responses of microglia in the pathogenesis of GC- mediated ELS-associated disorders such as schizophrenia, attention-deficit hyperactivity disorder and autism spectrum disorder.