Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia.

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.

Mitochondrial Targeted Cerium Oxide Nanoclusters for Radiation Protection and Promoting Hematopoiesis.

Purpose: Mitochondrial oxidative stress is an important factor in cell apoptosis. Cerium oxide nanomaterials show great potential for scavenging free radicals and simulating superoxide dismutase (SOD) and catalase (CAT) activities. To solve the problem of poor targeting of cerium oxide nanomaterials, we designed albumin-cerium oxide nanoclusters (TPP-PCNLs) that target the modification of mitochondria with triphenyl phosphate (TPP). TPP-PCNLs are expected to simulate the activity of superoxide dismutase, continuously remove reactive oxygen species, and play a lasting role in radiation protection. Methods: First, cerium dioxide nanoclusters (CNLs), polyethylene glycol cerium dioxide nanoclusters (PCNLs), and TPP-PCNLs were characterized in terms of their morphology and size, ultraviolet spectrum, dispersion stability and cellular uptake, and colocalization Subsequently, the anti-radiation effects of TPP-PCNLs were investigated using in vitro and in vivo experiments including cell viability, apoptosis, comet assays, histopathology, and dose reduction factor (DRF). Results: TPP-PCNLs exhibited good stability and biocompatibility. In vitro experiments indicated that TPP-PCNLs could not only target mitochondria excellently but also regulate reactive oxygen species (ROS)levels in whole cells. More importantly, TPP-PCNLs improved the integrity and functionality of mitochondria in irradiated L-02 cells, thereby indirectly eliminating the continuous damage to nuclear DNA caused by mitochondrial oxidative stress. TPP-PCNLs are mainly targeted to the liver, spleen, and other extramedullary hematopoietic organs with a radiation dose reduction factor of 1.30. In vivo experiments showed that TPP-PCNLs effectively improved the survival rate, weight change, hematopoietic function of irradiated animals. Western blot experiments have confirmed that TPP-PCNLs play a role in radiation protection by regulating the mitochondrial apoptotic pathway. Conclusion: TPP-PCNLs play a radiologically protective role by targeting extramedullary hematopoietic organ-liver cells and mitochondria to continuously clear ROS.

Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system.

In bone marrow transplantation (BMT), hematopoiesis-reconstituting cells are introduced following myeloablative treatment, which eradicates existing hematopoietic cells and disrupts stroma within the hematopoietic tissue. Both hematopoietic cells and stroma then undergo regeneration. Our study compares the outcomes of a second BMT administered to mice shortly after myeloablative treatment and the first BMT, with those of a second BMT administered to mice experiencing robust hematopoietic regeneration after the initial transplant. We evaluated the efficacy of the second BMT in terms of engraftment efficiency, types of generated blood cells, and longevity of function. Our findings show that regenerating hematopoiesis readily accommodates newly transplanted stem cells, including those endowed with a robust capacity for generating B and T cells. Importantly, our investigation uncovered a window for preferential engraftment of transplanted stem cells coinciding with the resumption of blood cell production. Repeated BMT could intensify hematopoiesis reconstitution and enable therapeutic administration of genetically modified autologous stem cells.

MYB: A Key Transcription Factor in the Hematopoietic System Subject to Many Levels of Control.

MYB is a master regulator and pioneer factor highly expressed in hematopoietic progenitor cells (HPCs) where it contributes to the reprogramming processes operating during hematopoietic development. MYB plays a complex role being involved in several lineages of the hematopoietic system. At the molecular level, the MYB gene is subject to intricate regulation at many levels through several enhancer and promoter elements, through transcriptional elongation control, as well as post-transcriptional regulation. The protein is modulated by post-translational modifications (PTMs) such as SUMOylation restricting the expression of its downstream targets. Together with a range of interaction partners, cooperating transcription factors (TFs) and epigenetic regulators, MYB orchestrates a fine-tuned symphony of genes expressed during various stages of haematopoiesis. At the same time, the complex MYB system is vulnerable, being a target for unbalanced control and cancer development.

Direct RNA sequencing of astronaut blood reveals spaceflight-associated m6A increases and hematopoietic transcriptional responses.

The advent of civilian spaceflight challenges scientists to precisely describe the effects of spaceflight on human physiology, particularly at the molecular and cellular level. Newer, nanopore-based sequencing technologies can quantitatively map changes in chemical structure and expression at single molecule resolution across entire isoforms. We perform long-read, direct RNA nanopore sequencing, as well as Ultima high-coverage RNA-sequencing, of whole blood sampled longitudinally from four SpaceX Inspiration4 astronauts at seven timepoints, spanning pre-flight, day of return, and post-flight recovery. We report key genetic pathways, including changes in erythrocyte regulation, stress induction, and immune changes affected by spaceflight. We also present the first m6A methylation profiles for a human space mission, suggesting a significant spike in m6A levels immediately post-flight. These data and results represent the first longitudinal long-read RNA profiles and RNA modification maps for each gene for astronauts, improving our understanding of the human transcriptome's dynamic response to spaceflight.

Standardized generation of human iPSC-derived hematopoietic organoids and macrophages utilizing a benchtop bioreactor platform under fully defined conditions.

BACKGROUND: There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac). METHODS: We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks. RESULTS: The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone. CONCLUSIONS: The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications.

Deficiency of glucocorticoid receptor in bone marrow adipocytes has mild effects on bone and hematopoiesis but does not influence expansion of marrow adiposity with caloric restriction.

Introduction: Unlike white adipose tissue depots, bone marrow adipose tissue (BMAT) expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unclear, prior research suggested an intermediary role for increased circulating glucocorticoids. Methods: In this study, we utilized a recently described mouse model (BMAd-Cre) to exclusively target bone marrow adipocytes (BMAds) for elimination of the glucocorticoid receptor (GR) (i.e. Nr3c1) whilst maintaining GR expression in other adipose depots. Results: Mice lacking GR in BMAds (BMAd-Nr3c1 -/-) and control mice (BMAd-Nr3c1 +/+) were fed ad libitum or placed on a 30% CR diet for six weeks. On a normal chow diet, tibiae of female BMAd-Nr3c1-/- mice had slightly elevated proximal trabecular metaphyseal bone volume fraction and thickness. Both control and BMAd-Nr3c1-/- mice had increased circulating glucocorticoids and elevated numbers of BMAds in the proximal tibia following CR. However, no significant differences in trabecular and cortical bone were observed, and quantification with osmium tetroxide and µCT revealed no difference in BMAT accumulation between control or BMAd-Nr3c1 -/- mice. Differences in BMAd size were not observed between BMAd-Nr3c1-/- and control mice. Interestingly, BMAd-Nr3c1-/- mice had decreased circulating white blood cell counts 4 h into the light cycle. Discussion: In conclusion, our data suggest that eliminating GR from BMAd has minor effects on bone and hematopoiesis, and does not impair BMAT accumulation during CR.

Hematologic Manifestations in Primary Mitochondrial Diseases.

Primary mitochondrial disorders (PMDs) are known for their pleiotropic manifestations in humans, affecting almost any organ or system at any time. Hematologic manifestations, such as cytopenias and sideroblastic anemia, occur in 10% to 30% of patients with confirmed PMDs. These can be the initial presenting features or complications that develop over time. Surveillance for these manifestations allows for prompt identification and treatment. This article provides an overview of the pathophysiology underpinning the hematologic effects of mitochondrial dysfunction, discussing the 3 key roles of the mitochondria in hematopoiesis: providing energy for cell differentiation and function, synthesizing heme, and generating iron-sulfur clusters. Subsequently, the diagnosis and management of mitochondrial disorders are discussed, focusing on hematologic manifestations and the specific conditions commonly associated with them. Through this, we aimed to provide a concise point of reference for those considering a mitochondrial cause for a patient's hematologic abnormality, or for those considering a hematologic manifestation in a patient with known or suspected mitochondrial disease.

From Marrow to Bone and Fat: Exploring the Multifaceted Roles of Leptin Receptor Positive Bone Marrow Mesenchymal Stromal Cells.

The bone marrow (BM) stromal cell microenvironment contains non-hematopoietic stromal cells called mesenchymal stromal cells (MSCs). MSCs are plastic adherent, form CFU-Fs, and give rise to osteogenic, adipogenic, chondrogenic progenitors, and most importantly provide HSC niche factor chemokine C-X-C motif ligand 12 (CXCL12) and stem cell factor (SCF). Different authors have defined different markers for mouse MSC identification like PDGFR+Sca-1+ subsets, Nestin+, or LepR+ cells. Of these, the LepR+ cells are the major source of SCF and CXCL12 in the BM microenvironment and play a major role in HSC maintenance and hematopoiesis. LepR+ cells give rise to most of the bones and BM adipocytes, further regulating the microenvironment. In adult BM, LepR+ cells are quiescent but after fracture or irradiation, they proliferate and differentiate into mesenchymal lineage osteogenic, adipogenic and/or chondrogenic cells. They also play a crucial role in the steady-state hematopoiesis process, as well as hematopoietic regeneration and the homing of hematopoietic stem cells (HSCs) after myeloablative injury and/or HSC transplantation. They line the sinusoidal cavities, maintain the trabeculae formation, and provide the space for HSC homing and retention. However, the LepR+ cell subset is heterogeneous; some subsets have higher adipogenic potential, while others express osteollineage-biased genes. Different transcription factors like Early B cell factor 3 (EBF3) or RunX2 help maintain this balance between the self-renewing and committed states, whether osteogenic or adipogenic. The study of LepR+ MSCs holds immense promise for advancing our understanding of HSC biology, tissue regeneration, metabolic disorders, and immune responses. In this review, we will discuss the origin of the BM resident LepR+ cells, different subtypes, and the role of LepR+ cells in maintaining hematopoiesis, osteogenesis, and BM adipogenesis following their multifaceted impact.

Nuclear miRNAs: Gene Regulation Activities.

MicroRNAs (miRNAs) are small non-coding RNAs which contribute to the regulation of many physiological and pathological processes. Conventionally, miRNAs perform their activity in the cytoplasm where they regulate gene expression by interacting in a sequence-specific manner with mature messenger RNAs. Recent studies point to the presence of mature miRNAs in the nucleus. This review summarizes current findings regarding the molecular activities of nuclear miRNAs. These molecules can regulate gene expression at the transcriptional level by directly binding DNA on the promoter or the enhancer of regulated genes. miRNAs recruit different protein complexes to these regions, resulting in activation or repression of transcription, through a number of molecular mechanisms. Hematopoiesis is presented as a paradigmatic biological process whereby nuclear miRNAs possess a relevant regulatory role. Nuclear miRNAs can influence gene expression by affecting nuclear mRNA processing and by regulating pri-miRNA maturation, thus impacting the biogenesis of miRNAs themselves. Overall, nuclear miRNAs are biologically active molecules that can be critical for the fine tuning of gene expression and deserve further studies in a number of physiological and pathological conditions.

CellRank 2: unified fate mapping in multiview single-cell data.

Single-cell RNA sequencing allows us to model cellular state dynamics and fate decisions using expression similarity or RNA velocity to reconstruct state-change trajectories; however, trajectory inference does not incorporate valuable time point information or utilize additional modalities, whereas methods that address these different data views cannot be combined or do not scale. Here we present CellRank 2, a versatile and scalable framework to study cellular fate using multiview single-cell data of up to millions of cells in a unified fashion. CellRank 2 consistently recovers terminal states and fate probabilities across data modalities in human hematopoiesis and endodermal development. Our framework also allows combining transitions within and across experimental time points, a feature we use to recover genes promoting medullary thymic epithelial cell formation during pharyngeal endoderm development. Moreover, we enable estimating cell-specific transcription and degradation rates from metabolic-labeling data, which we apply to an intestinal organoid system to delineate differentiation trajectories and pinpoint regulatory strategies.

Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting.

Haematopoiesis dysregulation with the presence of immature myeloid and erythroid immunosuppressive cells are key characteristics of the immune escape phase of tumour development. Here, the role of in vitro generated B16F10 tumour cell-derived extracellular vesicles (tEVs) as indirect cellular communicators, participating in tumour-induced dysregulation of haematopoiesis, was explored. The isolated tEVs displayed features of small EVs with a size range of 100-200 nm, expressed the common EV markers CD63, CD9, and Alix, and had a spherical shape with a lipid bilayer membrane. Proteomic profiling revealed significant levels of angiogenic factors, particularly vascular endothelial growth factor (VEGF), osteopontin, and tissue factor, associated with the tEVs. Systemic administration of these tEVs in syngeneic mice induced splenomegaly and disrupted haematopoiesis, leading to extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and the development of anaemia. These effects closely mirrored those observed in tumour-bearing mice and were not seen after heat inactivating the tEVs. In vitro studies demonstrated that tEVs independently induced the expansion of bone marrow granulocytic myeloid suppressor cells and B cells while reducing the frequency of cells in the erythropoietic lineage. These effects of tEVs were significantly abrogated by the blockade of VEGF or heat inactivation. Our findings underscore the important role of tEVs in dysregulating haematopoiesis during the immune escape phase of cancer immunoediting, suggesting their potential as targets for addressing immune evasion and reinstating normal hematopoietic processes.

Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

Transcriptional Regulation of the Lineage-Determining Gene PU.1 in Normal and Malignant Hematopoiesis: Current Understanding and Therapeutic Perspective.

The ETS transcription factor PU.1 plays an essential role in blood cell development. Its precise expression pattern is governed by cis-regulatory elements (CRE) acting at the chromatin level. CREs mediate the fine-tuning of graded levels of PU.1, deviations of which can cause acute myeloid leukemia. In this review, we perform an in-depth analysis of the regulation of PU.1 expression in normal and malignant hematopoiesis. We elaborate on the role of trans-acting factors and the biomolecular interplays in mediating local chromatin dynamics. Moreover, we discuss the current understanding of CRE bifunctionality exhibiting enhancer or silencer activities in different blood cell lineages and future directions toward gene-specific chromatin-targeted therapeutic development.

BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells.

Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

B Cells of Early-life Origin Defined by RAG2-based Lymphoid Cell Tracking under Native Hematopoietic Conditions.

During the perinatal period, the immune system sets the threshold to select either response or tolerance to environmental Ags, which leads to the potential to provide a lifetime of protection and health. B-1a B cells have been demonstrated to develop during this perinatal time window, showing a unique and restricted BCR repertoire, and these cells play a major role in natural Ab secretion and immune regulation. In the current study, we developed a highly efficient temporally controllable RAG2-based lymphoid lineage cell labeling and tracking system and applied this system to understand the biological properties and contribution of B-1a cells generated at distinct developmental periods to the adult B-1a compartments. This approach revealed that B-1a cells with a history of RAG2 expression during the embryonic and neonatal periods dominate the adult B-1a compartment, including those in the bone marrow (BM), peritoneal cavity, and spleen. Moreover, the BCR repertoire of B-1a cells with a history of RAG2 expression during the embryonic period was restricted, becoming gradually more diverse during the neonatal period, and then heterogeneous at the adult stage. Furthermore, more than half of plasmablasts/plasma cells in the adult BM had embryonic and neonatal RAG2 expression histories. Moreover, BCR analysis revealed a high relatedness between BM plasmablasts/plasma cells and B-1a cells derived from embryonic and neonatal periods, suggesting that these cell types have a common origin. Taken together, these findings define, under native hematopoietic conditions, the importance in adulthood of B-1a cells generated during the perinatal period.

A newly identified gene Ahed plays essential roles in murine haematopoiesis.

The development of haematopoiesis involves the coordinated action of numerous genes, some of which are implicated in haematological malignancies. However, the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here, we report a previously uncharacterised gene in haematopoiesis, identified by screening mutant embryonic stem cells. The gene, 'attenuated haematopoietic development (Ahed)', encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward, leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed-deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model, we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways, and published databases present cancer patients with somatic mutations in Ahed. Collectively, our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis, implicating its association with malignancies.

Systematic single-cell analysis reveals dynamic control of transposable element activity orchestrating the endothelial-to-hematopoietic transition.

BACKGROUND: The endothelial-to-hematopoietic transition (EHT) process during definitive hematopoiesis is highly conserved in vertebrates. Stage-specific expression of transposable elements (TEs) has been detected during zebrafish EHT and may promote hematopoietic stem cell (HSC) formation by activating inflammatory signaling. However, little is known about how TEs contribute to the EHT process in human and mouse. RESULTS: We reconstructed the single-cell EHT trajectories of human and mouse and resolved the dynamic expression patterns of TEs during EHT. Most TEs presented a transient co-upregulation pattern along the conserved EHT trajectories, coinciding with the temporal relaxation of epigenetic silencing systems. TE products can be sensed by multiple pattern recognition receptors, triggering inflammatory signaling to facilitate HSC emergence. Interestingly, we observed that hypoxia-related signals were enriched in cells with higher TE expression. Furthermore, we constructed the hematopoietic cis-regulatory network of accessible TEs and identified potential TE-derived enhancers that may boost the expression of specific EHT marker genes. CONCLUSIONS: Our study provides a systematic vision of how TEs are dynamically controlled to promote the hematopoietic fate decisions through transcriptional and cis-regulatory networks, and pre-train the immunity of nascent HSCs.