Human Hematopoietic Development Research Articles

Identification and characterization of human hematopoietic mesoderm.

The embryonic mesoderm comprises heterogeneous cell subpopulations with distinct lineage biases. It is unclear whether a bias for the human hematopoietic lineage emerges at this early developmental stage. In this study, we integrated single-cell transcriptomic analyses of human mesoderm cells from embryonic stem cells and embryos, enabling us to identify and define the molecular features of human hematopoietic mesoderm (HM) cells biased towards hematopoietic lineages. We discovered that BMP4 plays an essential role in HM specification and can serve as a marker for HM cells. Mechanistically, BMP4 acts as a downstream target of HDAC1, which modulates the expression of BMP4 by deacetylating its enhancer. Inhibition of HDAC significantly enhances HM specification and promotes subsequent hematopoietic cell differentiation. In conclusion, our study identifies human HM cells and describes new mechanisms for human hematopoietic development.

SETD7 promotes lateral plate mesoderm formation by modulating the Wnt/β-catenin signaling pathway

The role of SET domain containing 7 (SETD7) during human hematopoietic development remains elusive. Here, we found that deletion of SETD7 attenuated the generation of hematopoietic progenitor cells (HPCs) during the induction of hematopoietic differentiation from human embryonic stem cells (hESCs). Further analysis specified that SETD7 was required for lateral plate mesoderm (LPM) specification but dispensable for the generation of endothelial progenitor cells (EPCs) and HPCs. Mechanistically, rather than depending on its histone methyltransferase activity, SETD7 interacted with β-catenin at lysine residue 180 facilitated its degradation. Diminished SETD7 expression led to the accumulation of β-catenin and the consequent activation of the Wnt signaling pathway, which altered LPM patterning and facilitated the production of paraxial mesoderm (PM). Taken together, the findings indicate that SETD7 is related to LPM and PM patterning via posttranslational regulation of the Wnt/β-catenin signaling pathway, providing novel insights into mesoderm specification during hematopoietic differentiation from hESCs.

Generation of RUNX1c-eGFP induced pluripotent stem cell, MUSIi012-A-4, using CRISPR/Cas9

Runt-Related Transcription Factor 1c (RUNX1c) plays an important role in regulating the development of hematopoietic stem cells (HSC). Using CRISPR/Cas9 gene editing technology, we established a RUNX1c-eGFP reporter cell line from the MUSIi012-A cell line. The MUSIi012-A-4 cell line has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro and in vivo. This cell line serves as a valuable model to observe the expression of RUNX1c via eGFP tracking during human hematopoietic development.

CD1d expression demarcates CDX4+ hemogenic mesoderm with definitive hematopoietic potential

To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.

Identification of a retinoic acid-dependent haemogenic endothelial progenitor from human pluripotent stem cells.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. During embryonic development, HSCs derive from haemogenic endothelium (HE) in a NOTCH- and retinoic acid (RA)-dependent manner. Although a WNT-dependent (WNTd) patterning of nascent hPSC mesoderm specifies clonally multipotent intra-embryonic-like HOXA+ definitive HE, this HE is functionally unresponsive to RA. Here we show that WNTd mesoderm, before HE specification, is actually composed of two distinct KDR+ CD34neg populations. CXCR4negCYP26A1+ mesoderm gives rise to HOXA+ multilineage definitive HE in an RA-independent manner, whereas CXCR4+ ALDH1A2+ mesoderm gives rise to HOXA+ multilineage definitive HE in a stage-specific, RA-dependent manner. Furthermore, both RA-independent (RAi) and RA-dependent (RAd) HE harbour transcriptional similarity to distinct populations found in the early human embryo, including HSC-competent HE. This revised model of human haematopoietic development provides essential resolution to the regulation and origins of the multiple waves of haematopoiesis. These insights provide the basis for the generation of specific haematopoietic populations, including the de novo specification of HSCs.

Generation of a GFI1-flag knock-in human embryonic stem cell line using CRISPR-Cas9 technology

GFI1 is a DNA binding transcriptional repressor, it is shown to be an important gene associated with blood cells development and many blood diseases (Möröy et al., 2015). But the role of GFI1 in human hematopoieticdevelopment has not been known (Thambyrajah et al., 2016). To illustrate the function of GFI1 in human hematopoieticdevelopment, we constructed a GFI1-2 × flag-tag knock-in human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting, and it would be the effective tool to study GFI1. The cell line could express GFI1-2 × flag-tag and can be identified with western blot and immunofluorescence. This cell line maintains stem cell morphology, and displays normal karyotype, pluripotent stem cell marker expression and differentiation potential.

Are Induced Pluripotent Stem Cells a Step towards Modeling Pediatric Leukemias?

Despite enormous improvements in pre-clinical and clinical research, acute leukemia still represents an open challenge for pediatric hematologists; both for a significant relapse rate and for long term therapy-related sequelae. In this context, the use of an innovative technology, such as induced pluripotent stem cells (iPSCs), allows to finely reproduce the primary features of the malignancy and can be exploited as a model to study the onset and development of leukemia in vitro. The aim of this review is to explore the recent literature describing iPSCs as a key tool to study different types of hematological malignancies, comprising acute myeloid leukemia, non-down syndrome acute megakaryoblastic leukemia, B cell acute lymphoblastic leukemia, and juvenile myelomonocytic leukemia. This model demonstrates a positive impact on pediatric hematological diseases, especially in those affecting infants whose onsets is found in fetal hematopoiesis. This evidence highlights the importance of achieving an in vitro representation of the human embryonic hematopoietic development and timing-specific modifications, either genetic or epigenetic. Moreover, further insights into clonal evolution studies shed light in the way of a new precision medicine era, where patient-oriented decisions and therapies could further improve the outcome of pediatric cases. Nonetheless, we will also discuss here the difficulties and limitations of this model.

P53 Is a Crucial Regulator of Hemogenic Endothelial Cell Differentiation from Human Induced Pluripotent Stem Cells

De novo generation of hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (hiPSCs) could provide a virtually unlimited supply of autologous HSCs for clinical transplantation, and offer various approaches that enable gene therapy, drug discovery, disease modeling, and in vitro modeling of human hematopoietic development. However, the derivation of long-term self-renewing HSCs from hiPSCs in culture remains elusive. The tumor suppressor protein p53 plays important roles in normal and malignant hematopoiesis, and Trp53-deficient mice exhibit increased number of HSCs. Although activation of p53 is known to promote differentiation of hPSCs and hPSCs recurrently acquire TP53 dominant negative mutations, its role in hematopoietic differentiation of hiPSCs has not been explored.To differentiate hiPSCs into hematopoietic stem and progenitor cells (HSPCs), we used embryoid body (EB) formation method to first differentiate hiPSCs into hemogenic endothelial (HE) cells that express the CD34 highCD144 +CD73 -CD184 -CD43 -CD235a - cell-surface markers. HE cells were then transferred onto a Matrigel-coated plate to undergo endothelial-to-hematopoietic transition (EHT) to generate HSPCs that express the CD34 midCD45 mid cell-surface markers. Developed HSPCs were functionally evaluated by colony forming assay.We observed that the expression of CDKN1A, a p53 target gene, was upregulated in hiPSC-derived EBs and HSPCs over the course of differentiation. To investigate the role of p53 in the generation of HSPCs from hiPSCs, we genetically deleted TP53 in hiPSCs followed by hematopoietic differentiation. While TP53 deletion increased the growth of EBs, it resulted in severe impairment of differentiation into HE cells and overall production of HSPCs that can form colonies. During HE differentiation from hiPSCs, TP53-deficient EBs showed significant reduction of endothelial-lineage gene expression, such as ETV2, CDH5, and PECAM1, as well as expression of RUNX1, a master transcription factor required for HE specification. These results indicate the indispensable role of p53 in HE differentiation from hiPSCs.We then examined the effect of p53 activation on HE differentiation from hiPSCs by pharmacological activation of p53 in hiPSC-derived cells. Transient activation of p53 by Nutlin-3, a small molecule that inhibits the p53-HDM2 interaction and protects p53 from proteasomal degradation, only during HE differentiation but not during EHT significantly promoted HSPC generation as compared to the vehicle treated control.Our findings shed light on the importance of selecting hiPSC lines that retain normal p53 activity for HE differentiation, and provide an approach to promote hematopoietic differentiation of hiPSCs by transiently activating p53 during HE differentiation. DisclosuresKanaujiya: Synthego: Other: Scientific Advisory; eGenesis: Other: Scientific Advisory.

Recapitulative haematopoietic development of human pluripotent stem cells in the absence of exogenous haematopoietic cytokines.

To improve the recapitulative quality of human pluripotent stem cell (hPSC) differentiation, we removed exogenous haematopoietic cytokines from the defined differentiation system. Here, we show that endogenous stimuli and VEGF are sufficient to induce robust hPSC‐derived haematopoiesis, intensive generation of haematopoietic progenitors, maturation of blood cells and the emergence of definitive precursor cells including those that phenotypically identical to early human embryonic haematopoietic stem cells (HSCs). Moreover, the cytokine‐free system produces significantly higher numbers of haematopoietic progenitors compared to the published protocols. The removal of cytokines revealed a broad developmental potential of the early blood cells, stabilized the hPSC‐derived definitive precursors and led to spontaneous activation of inflammatory signalling. Our cytokine‐free protocol is simple, efficient, reproducible and applicable for embryonic stem cells (ESCs) and induced PSCs. The spectrum of recapitulative features of the novel protocol makes the cytokine‐free differentiation a preferred model for studying the early human haematopoietic development.

Global Analysis of Gene Networks Involved in Hematopoiesis.

The hematopoietic system consists of more than ten differentiated cell types, which are derived from a single type of hematopoietic stem cell. Conceptually and experimentally, the determination of lineage choice, and the plasticity, extent, and robustness of lineage specific patterns in gene expression are current topics of intensive interest and investigation. To approach to these issues, we compared global analyses of a large number of genes in more than 10 primary cells cell types including stem cells, myeloid cells, erythroid cells and lymphocytes as well as certain cell lines.We are carrying out this global work on multiple different platforms in parallel:Gene expression. For these analyses we have used Affymetrix oligonucleoide arrays (U133) for cDNA quantitation, Affymetrix oligionucleotide arrays covering the ENCODE regions of the human genome, a PCR fragment Chromosome 22.2 genomic tiling array and a PCR fragment promoter array. U133 chip analyses were performed on the RNA samples extracted from all the cell types in this study. PCA analysis of gene expression patterns of these cell types shows that there is a pervasive lineage specific pattern of expression of most genes, and this lineage difference accounts for a substantial fraction of the total variation in gene expression between samples. Our data provides a guide for investigating the transcriptional control of both major lineage specific and broadly expressed genes for many hematopoietic cell types.DNA-Protein interaction. More than 100 transcription factors (TFs) were studied by western-blotting, using NB4 promyelocytic cells, neutrophils or monocytes differentiated from NB4 cells, primary human PMNs, K562 and Hela cells. Our result showed that many TFs are myeloid specific, their protein expression level are very different from the mRNA level of mature cells. To further study the regulatory mechanisms, chromatin-IP analysis has been done on microarrays (Chip-chip). Immunoprecipitaiton studies of NF-KB, and CEBPE were carried out with chromosome 22 genomic tiling arrays and promoter arrays. Many gene targets of these two TFs were discovered. Importantly, we found that these two TFs not only bind to the promoter regions of their target gene, but also the introns and inter-genic sequences. This, and related results from other groups, shows an unexpected complexity in transcriptional control mechanisms.Proteomics. ICAT mass spectroscopy and DIGE have been used for protein expression analysis of 6 cell types. These studies also have generated a large amount of data, which are being analyzed.Our analyses are producing a wealth of information on the molecular anatomy of human hematopoietic development, especially myeloid differentiation. We are exploring the construction of network maps of hematopoiesis based on these data.

Vitronectin-activated αvβ3 and αvβ5 integrin signalling specifies haematopoietic fate in human pluripotent stem cells.

ObjectivesVitronectin (VTN) has been widely used for the maintenance and expansion of human pluripotent stem cells (hPSCs) as feeder‐free conditions. However, the effect of VTN on hPSC differentiation remains unclear. Here, we investigated the role of VTN in early haematopoietic development of hPSCs.Materials and MethodsA chemically defined monolayer system was applied to study the role of different matrix or basement membrane proteins in haematopoietic development of hPSCs. The role of integrin signalling in VTN‐mediated haematopoietic differentiation was investigated by integrin antagonists. Finally, small interfering RNA was used to knock down integrin gene expression in differentiated cells.ResultsWe found that the haematopoietic differentiation of hPSCs on VTN was far more efficient than that on Matrigel that is also often used for hPSC culture. VTN promoted the fate determination of endothelial‐haematopoietic lineage during mesoderm development to generate haemogenic endothelium (HE). Moreover, we demonstrated that the signals through αvβ3 and αvβ5 integrins were required for VTN‐promoted haematopoietic differentiation. Blocking αvβ3 and αvβ5 integrins by the integrin antagonists impaired the development of HE, but not endothelial‐to‐haematopoietic transition (EHT). Finally, both αvβ3 and αvβ5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of αv, β3 or β5.ConclusionThe established VTN‐based monolayer system of haematopoietic differentiation of hPSCs presents a valuable platform for further investigating niche signals involved in human haematopoietic development.

Selective antisense oligonucleotide inhibition of human IRF4 prevents malignant myeloma regeneration via cell cycle disruption.

In multiple myeloma, inflammatory and anti-viral pathways promote disease progression and cancer stem cell generation. Using diverse pre-clinical models, we investigated the role of interferon regulatory factor 4 (IRF4) in myeloma progenitor regeneration. In a patient-derived xenograft model that recapitulates IRF4 pathway activation in human myeloma, we test the effects of IRF4 antisense oligonucleotides (ASOs) and identify a lead agent for clinical development (ION251). IRF4 overexpression expands myeloma progenitors, while IRF4 ASOs impair myeloma cell survival and reduce IRF4 and c-MYC expression. IRF4 ASO monotherapy impedes tumor formation and myeloma dissemination in xenograft models, improving animal survival. Moreover, IRF4 ASOs eradicate myeloma progenitors and malignant plasma cells while sparing normal human hematopoietic stem cell development. Mechanistically, IRF4 inhibition disrupts cell cycle progression, downregulates stem cell and cell adhesion transcript expression, and promotes sensitivity to myeloma drugs. These findings will enable rapid clinical development of selective IRF4 inhibitors to prevent myeloma progenitor-driven relapse.

High-Efficiency Differentiation of Human Pluripotent Stem Cells to Hematopoietic Stem/Progenitor Cells in Random Positioning Machine Bioreactors.

Human pluripotent stem cells (PSCs) are known to differentiate into almost all the blood lineage cells in vitro and hold a great promise for studying human early hematopoietic development and have a huge potential in the treatment of hematological disorders. Although several methods of hematopoietic stem/progenitor cell (HSPC) differentiation have been developed, the HSPC yields achieved using these strategies are not yet available for clinical application. Recently, bioreactor-based devices and biochemical factors synergistically have been used to induce hematopoietic differentiation and showed a potential role in hematopoiesis. This chapter describes a protocol for using a random positioning machine bioreactor to culture human PSCs and the large-scale production of HPCs. Techniques for characterizing the differentiated cells and assessing the efficiency of hematopoietic differentiation in the bioreactor with immunostaining and flow cytometry are also presented.

Generation of Retinoic Acid-Dependent Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a− mesodermal population that gives rise to a clonally multipotent HOXA+ definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes and is functionally unresponsive to RA treatment. Thus, the specification of an RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNA-seq (scRNA-seq) analyses, we identified that 2 distinct KDR+CD235a− populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, KDR+CD235a−CXCR4− mesoderm expressed CYP26A1, an RA degrading enzyme, and harbored definitive hematopoietic potential within hPSC differentiation cultures in the absence of RA signaling, indicating the HE specified from CXCR4− mesoderm as RA-independent (RAi). In sharp contrast, KDR+CD235a−CXCR4+ mesoderm exclusively expressed ALDH1A2, the key enzyme in the synthesis of RA, but lacked hematopoietic potential under the same culture conditions. However, the stage-specific application of RA signaling to CXCR4+ mesoderm resulted in the robust specification of CD34+HOXA+ HE with definitive erythroid, myeloid, and lymphoid hematopoietic potential, establishing this HE as RA-dependent (RAd). Furthermore, while RAi HE entirely failed to persist following murine hematopoietic xenografts, RAd HE transiently persisted within the peripheral blood and bone marrow of murine hosts. To assess whether these functionally distinct hPSC mesodermal progenitors are physiologically relevant to human embryonic development, we integrated scRNA-seq datasets from the hPSC mesodermal cultures and a gastrulating human embryo. These analyses revealed that in vivo, distinct KDR+CXCR4−CYP26A1+ and KDR+CXCR4+ALDH1A2+ populations can be found at the stage of emergent mesoderm, following patterning of nascent mesoderm. Additional comparison to later stage human embryos demonstrated that RAd HE has a more fetal-like HOXA expression pattern than RAi HE. Scoring of single fetal HE cells against hPSC-derived HE revealed that while some early fetal HE cells were similar to RAi HE, the late fetal HE cells, which are hypothesized to give rise to HSCs, were more similar to RAd HE. Lastly, as HSC-competent HE is expected to express arterial genes, we found a subset of late fetal HE with this phenotype that were exclusively similar to RAd HE. Collectively, these data represent the first ever characterization of RA-dependent hPSC-derived definitive hematopoiesis and its mesodermal progenitor. Additionally, we provide evidence for in vivo mesodermal and HE correlates for both RAi and RAd hematopoietic programs within human embryos. This novel insight into human hematopoietic development will serve as an important tool for modeling development and ultimately provide the basis for de novo specification of HSCs. Disclosures No relevant conflicts of interest to declare.

Transcriptional, Protein-Level and Functional Profiling of Human Fetal Liver (FL)-Derived Hematopoietic Stem Cells (HSCs) at Single Cell Resolution

Intro: The complex and tightly regulated process of human hematopoietic development culminates in the production of hematopoietic stem cells (HSCs), which subsequently acquire functional competence and undergo expansion in the fetal liver (FL). The establishment of a high-resolution molecular signature of FL HSCs provides insights into HSC biology with potential utility in the purification and expansion of engraftable HSCs ex vivo and the generation of HSCs from pluripotent cell sources. To profile HSCs at this developmental stage, we performed CITE-Seq, a technique that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies to simultaneously map transcriptional and protein-level expression in individual cells. To connect expression profiles with functional engraftment, we have coupled this with transplantation assays in immunocompromised mice. Methods: In these studies, three populations of human FL cells were used: CD34- cells, CD34+ cells and CD34+ cells furtherenrichedby expression of GPI-80, a marker tightly linked to engraftment potential, to explicitly identify HSCs capable of long-term engraftment. These populations were stained with a panel of oligo-tagged antibodies, processed via the 10X Genomics platform, and sequenced (26,407 total cells). Results: Transplantation experiments using the same sorted fractions that were assayed by CITE-seq revealed superior engraftment potential of the GPI-80+ fraction, and thus enrichment for bona fide HSCs at the functional level. This functional signature coincided with enrichment for known HSC markers such as ITGA6 (CD49f), PROCR (EPCR), CD164, MLLT3, HLF, CLEC9A and HMGA2 at the transcriptional level. As such, by profiling >7000 GPI-80+ cells, we have achieved unprecedented resolution of the engraftable HSC compartment within the FL. Combined analysis of all captured FL fractions accurately recapitulated the hematopoietic landscape of the FL at this developmental stage, representing the expected hematopoietic lineages and cell types. To gain further insight into FL HSCs, we next focused on the CD34+ HSC/progenitor compartment where we tracked cluster dynamics upon functional HSC enrichment between the CD34+ bulk and GPI-80+ sample. We noted a prominent (4-fold) increase in a cluster marked by enrichment for genes including RGCC, LMNA, VIM, ID1 and ID3 as well as components of the AHR pathway, suggesting that this expression profile strongly correlates with engraftment potential. Notably, LMNA is expressed in postnatal HSCs and its expression has been shown to decrease upon aging (Grigoryan et al., 2018). In line with this, we also found that LMNA is more highly expressed in our prenatal FL HSPCs compared to postnatal HSPCs. This data suggests a potential role for LMNA in endowing FL HSCs with their superior engraftment potential compared to postnatal HSCs. To complement our transcriptomic characterization of engraftment potential, we also collected cell surface marker expression data based on sequencing of a series of antibody-derived tags (ADTs). This additional layer of information uncovered expression patterns that weren't readily apparent based on mRNA expression data and inspired us to use this ADT information to gate out populations of interest via in silico sorting. This enabled us to compare the transcriptional profiles associated with well-described HSC enrichment signatures to assess whether they represent equivalent cell populations. We compared CD34+CD90+CD49f+ (~Notta et al., 2011) vs CD34+CD133+GPI-80+ (~Sumide et al., 2018) vs CD34+EPCR+ (~Subramaniam et al., 2019) in silico sorted cells and found a strong overlap in enriched genes, suggesting that the transcriptomic signature corresponding to engraftment potential identified in this work is not exclusive to GPI-80+ sorted cells but represents engraftable HSCs beyond this enrichment strategy. Conclusion: We have achieved unprecedented resolution of the engraftable HSC compartment in the human FL. Combining transcriptional profiling of the engraftment potential of FL HSCs with cell surface level expression data provides an in-depth characterization of this unique and developmentally relevant HSC source. This data will be valuable in optimizing the purification and expansion of engraftable HSCs ex vivo as well as in guiding the in vitro generation of HSCs from pluripotent cell sources. Disclosures No relevant conflicts of interest to declare.

Single Cell Resolution Mapping of Hematopoietic Stem and Progenitor Cell States throughout Human Life

The hematopoietic system continuously generates and replenishes the supply of circulating blood cells from embryonic life throughout the entirety of human lifespan. Studies in mouse development have shown that the repertoire of mature blood cell types produced changes dramatically during development and aging, with hematopoietic stem and progenitor cells (HSPCs) adapting their output to meet age-specific physiologic needs. In humans, it is presumed that age-dependent changes in the production of mature blood lineages underlie the tendency of blood disorders to skew toward certain ages of onset. The observations that mature cell output changes throughout life but mechanisms of terminal hematopoietic differentiation within each lineage remain consistent suggest that age-specific hematopoietic states are programmed at the level of HSPCs. Although the developmental changes occurring in mouse hematopoiesis are well documented, the specific changes in human HSPC ontogeny occurring during prenatal development and postnatal aging from newborn, through childhood, and into adulthood are completely unknown. We hypothesized that temporal changes in human hematopoiesis are mediated by age-specific, occasionally transient, HSPC states and that mechanisms of HSPC lineage commitment change over time in order to meet the changing physiologic demands of the developing and aging human. To test this hypothesis, we comprehensively profiled human HSPC cell states from human fetal hematopoiesis through adulthood using single cell RNA sequencing (scRNAseq). We obtained CD34+ HSPCs from 14 different human donors covering a range of ages from first and second trimester fetal liver, umbilical cord blood, and pediatric and adult bone marrow. We obtained high quality sequencing data on a total of 38,873 individual HSPCs after filtering out apoptotic cells, hepatocytes, stromal cells, and endothelial cells. We then identified differentially expressed genes and performed dimensionality reduction and uniform manifold approximation and projection followed by Louvain clustering to identify 32 distinct cell types encompassing primitive stem and multipotent progenitor cells as well as committed progenitors in the myeloid, erythroid and lymphoid lineages. We found age-specific alterations in hematopoietic differentiation trajectories, particularly in the myeloid lineages. Additionally, we discovered an HSC, emerging in mid-gestation and diminishing at birth, with a characteristic immunophenotype and megakaryocyte (Meg) differentiation bias. Differential gene expression analysis of the Meg-biased fetal HSC identified increased expression of the MYB transcription factor relative to other HSCs, potentially illuminating a mechanistic role for MYB in driving megakaryocyte-erythroid progenitor (MEP) differentiation of fetal HSCs, that is distinct from the role of MYB in conferring erythroid differentiation bias at the MEP stage itself. Finally, we used this atlas of human developmental hematopoiesis to map lineage commitment and progenitor states in leukemia, highlighting the translational applicability of this resource. In summary, we have compiled the first comprehensive atlas of HSPCs across human development and aging. This resource allowed us to identify age-specific differentiation trajectories in human hematopoiesis and enabled identification of a Meg-biased fetal-specific HSC. Our research reveals novel mechanisms of maturation and aging of the human hematopoietic system, uncovers transient HSPC states and differentiation trajectories, and establishes a framework for interrogating the differentiation and maturation states of human leukemias that can likely be applied to other blood diseases. As a resource, we expect that this atlas will broadly impact the study of human hematopoietic development and aging, developmental immunology, and the pathophysiology of age-biased blood diseases. Disclosures No relevant conflicts of interest to declare.

Multi-layered Spatial Transcriptomics Identify Secretory Factors Promoting Human Hematopoietic Stem Cell Development.

SummaryHematopoietic stem cells (HSCs) first emerge in the embryonic aorta-gonad-mesonephros (AGM) region. Studies of model organisms defined intersecting signaling pathways that converge to promote HSC emergence predominantly in the ventral domain of the dorsal aorta. Much less is known about mechanisms driving HSC development in humans. Here, to identify secreted signals underlying human HSC development, we combined spatial transcriptomics analysis of dorsoventral polarized signaling in the aorta with gene expression profiling of sorted cell populations and single cells. Our analysis revealed a subset of aortic endothelial cells with a downregulated arterial signature and a predicted lineage relationship with the emerging HSC/progenitor population. Analysis of the ventrally polarized molecular landscape identified endothelin 1 as an important secreted regulator of human HSC development. The obtained gene expression datasets will inform future studies on mechanisms of HSC development in vivo and on generation of clinically relevant HSCs in vitro.

Biphasic Regulation of Mesenchymal Genes Controls Fate Switches During Hematopoietic Differentiation of Human Pluripotent Stem Cells.

Epithelial‐mesenchymal transition (EMT) or its reverse process mesenchymal‐epithelial transition (MET) occurs in multiple physiological and pathological processes. However, whether an entire EMT–MET process exists and the potential function during human hematopoiesis remain largely elusive. Utilizing human pluripotent stem cell (hPSC)‐based systems, it is discovered that while EMT occurs at the onset of human hematopoietic differentiation, MET is not detected subsequently during differentiation. Instead, a biphasic activation of mesenchymal genes during hematopoietic differentiation of hPSCs is observed. The expression of mesenchymal genes is upregulated during the fate switch from pluripotency to the mesoderm, sustained at the hemogenic endothelium (HE) stage, and attenuated during hemogenic endothelial cell (HEP) differentiation to hematopoietic progenitor cells (HPCs). A similar expression pattern of mesenchymal genes is also observed during human and murine hematopoietic development in vivo. Wnt signaling and its downstream gene SNAI1 mediate the up‐regulation of mesenchymal genes and initiation of mesoderm induction from pluripotency. Inhibition of transforming growth factor‐β (TGF‐β) signaling and downregulation of HAND1, a downstream gene of TGF‐β, are required for the downregulation of mesenchymal genes and the capacity of HEPs to generate HPCs. These results suggest that the biphasic regulation of mesenchymal genes is an essential mechanism during human hematopoiesis.

3022 – GENERATION OF RETINOIC ACID-DEPENDENT HEMOGENIC ENDOTHELIAL PROGENITORS FROM HUMAN PLURIPOTENT STEM CELLS

The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a- mesodermal population that gives rise to a clonally multipotent definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes, and is functionally unresponsive to RA treatment. Thus, the specification of a RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNAseq analyses, we identified that two distinct KDR+CD235a- populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, CYP26A1, a RA degrading enzyme, was expressed in KDR+CD235a-CXCR4- mesoderm, and this was the only mesodermal population harboring definitive hematopoietic potential. In sharp contrast, KDR+CD235a-CXCR4+ mesoderm expressed ALDH1A2, a key enzyme in the synthesis of RA and lacked any hematopoietic potential. However, the stage-specific application of retinoic acid signaling to this CXCR4+ mesodermal population resulted in the robust specification of CD34+ HE with multi-lineage definitive erythroid, myeloid and lymphoid hematopoietic potential. Further, in vivo correlates to these functionally-distinct mesodermal precursors could be found within a scRNA-seq dataset of gastrulating murine embryos. Additional comparison to human embryos demonstrated that CD34+ cells specified with retinol from CXCR4+ mesoderm have fetal-like HOXA expression. Collectively, this represents the first ever characterization of stage-specific RA-dependent hPSC-derived definitive hematopoiesis, and its mesodermal progenitor. This novel insight into human hematopoietic development will ultimately provide the basis for the de novo specification of HSCs.

3139 – TRANSCRIPTIONAL, PROTEIN-LEVEL AND FUNCTIONAL PROFILING OF HUMAN FETAL LIVER-DERIVED HEMATOPOIETIC STEM CELLS AT SINGLE CELL RESOLUTION

The complex and tightly regulated process of human hematopoietic development culminates in the production of hematopoietic stem cells (HSCs), which subsequently undergo expansion in the fetal liver (FL). The establishment of a high-resolution molecular signature of FL-HSCs would provide insights into HSC biology with potential utility in the purification and expansion of engraftable HSCs ex vivo and the generation of HSCs from pluripotent cell sources. To profile HSCs at this developmental stage, we performed CITE-Seq, a technique that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies. To connect expression profiles with functional engraftment, we have coupled this with transplantation assays in immunocompromised mice. In these studies, three populations of human FL cells were used: CD34- cells, CD34+ cells and CD34+ cells further enriched by expression of GPI-80, a marker tightly linked to engraftment potential, to acquire additional resolution of HSCs capable of long-term engraftment. These populations were stained with a panel of oligo-tagged antibodies, processed via the 10X platform, and sequenced (26.582 total cells). Simultaneous transplantation experiments of these three populations revealed superior engraftment potential of the CD34+GPI-80+ fraction, and thus enrichment for bona fide HSCs at the functional level. This coincided with enrichment for known HSC markers such as ITGA6 (CD49f), PROCR (EPCR), CD164, MLLT3, HLF and HMGA2 at the transcriptional level. As such, by profiling profiling >7000 GPI-80+CD34+ cells, we have achieved unprecedented resolution of the engraftable HSC compartment within the FL. Studying the transcriptional profile in combination with protein level expression of key HSC-related genes, will allow for an accurate characterization of these cells and provide insights into the biology behind their remarkable engraftment potential.