Primitive Hematopoiesis Research Articles

Zebrafish gata2 Orthologs Are Necessary but Not Sufficient for Developmental Hematopoiesis

Germline mutations in the GATA2 gene have been identified in patients with a variety of hematological disorders unified by their with predisposition to myelodysplastic syndromes (MDS) and defective immunity. The GATA2 gene encodes a transcription factor that plays a major role in hematopoiesis. However, the precise effects of GATA2 haploinsufficiency in developmental hematopoiesis and the impact of this in MDS predisposition remain unknown. We have developed zebrafish mutants of gata2 to model this and demonstrate that heterozygosity results in defective primitive and definitive granulopoiesis.Zebrafish carry two orthologues to the mammalian GATA2 gene: gata2a and gata2b . gata2a and gata2b demonstrate distinct expression patterns. By 32 hours post fertilization (hpf), expression of gata2a is seen throughout the dorsal aorta (DA) and cardinal vein, primitive erythrocytes and the nervous system. In contrast, gata2b expression is restricted to the ventral DA (VDA) and branchiomotor neurons. We hypothesised that this division of expression may translate into a requirement for both ohnologs as necessary but not sufficient for normal developmental hematopoiesis.Homozygous gata2a mutants carrying an 11bp deletion (gata2aum27 ) have been previously described. These mutants have abnormal vascular development in the aorta resulting in defective circulation. We analysed developmental hematopoiesis in these mutants. gata2aum27/um27 and gata2aum27 /+ showed normal hemangioblasts, and primitive hematopoiesis before 18 somites. At 22hpf however myeloid transcription factor cebpa showed a dose dependent decrease in expression resulting from gata2a loss, while pu.1 expression remained normal. This suggests that Gata2a may be a direct regulator of cebpa in primitive myeloid cells, or that cebpa expression defines a sub-population of myeloid cells. We then assessed hematopoietic stem cells (HSCs) in gata2aum27 mutants. gata2aum27/um27 showed severely decreased or absent HSCs in the caudal hematopoietic tissue (CHT, fetal liver equivalent) at 52hpf, likely due to absent blood flow in the aorta. gata2aum27/+ mutants also showed a decrease in HSCs at this time point, however this decrease was transient, resolving by 72hpf (A). Further assessment of definitive hematopoiesis showed that gata2um27/um27 lacked mature granulocytes and thymocytes, in keeping with the observed HSC defect. By contrast myeloid cells in gata2aum27/+ at 4dpf (assessed by Sudan Black (SB) stain) were unaffected (B,C) . Interestingly lck-expressing thymocytes showed a range of phenotypes, from normal, through reduced and some with a complete absence of thymocytes in one of their thymii.We next used CRISPR to generate a gata2b mutant line. This line carries a 4bp deletion leading to a truncated protein lacking both zinc fingers (gata2bU5008 ). In contrast to gata2aum27/um27 , gata2bu5008/5008 have normal circulation and were viable beyond 5dpf. Loss of gata2b by morpholino knockdown results in absence of HSC, however gata2b5008/+ showed no difference in HSC numbers in the CHT at 60hpf. Myeloid cells were unaffected in gata2b5008 mutants by SB at 48hpf, however, by 4dpf gata2b5008 show a dose dependent reduction in myeloid cells (D). This suggests that Gata2b regulates myeloid cell proliferation or differentiation after the emergence of HSCs.We next studied haematopoiesis in crosses of gata2aum27/+ to gata2bu5008/+ . Quantification of granulocytes showed normal numbers at 48hpf in all resulting genotypes. By 4dpf gata2b5008/+ have a reduced number of SB expressing granulocytes regardless of their gata2a genotype, suggesting that Gata2a is dispensable for definitive myelopoiesis (E). Quantification of HSCs show HSC numbers are unaffected in gata2aum27/+ ; gata2bu5008/+ at 48hpf. Assessment of thymocytes by contrast shows a greater than additive effect on thymocyte numbers than in single mutants alone, suggesting that both Gata2a and Gata2b contribute to lymphoid development in an additive manner. Further characterisation of the effects of gata2aum27/+ ; gata2bu5008/+ in combination is ongoing.In conclusion our results support the notion at that haploinsufficient levels of Gata2a and Gata2b are necessary but not sufficient for normal developmental hematopoiesis and that combined heterozygous loss of these genes models the haploinsufficieny observed in patients in GATA2 haploinsufficient diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GATA2 Is Essential for Endothelial-to-Hematopoietic Transition but Does Not Affect Formation of Hemogenic Endothelium

The formation of blood cells from hemogenic endothelium (HE) is a key element of leading to establishment of the hematopoietic system during embryogenesis. It has become increasingly clear that HE represents a distinct lineage of vascular endothelium with a capacity to undergo endothelial-to-hematopoietic transition (EHT). However, the processes guiding HE specification and EHT are poorly understood. The GATA2 transcription factor is of particular interest since it is critical for development of the entire hematopoietic system including hematopoietic stem cells (HSCs) during embryogenesis. Although GATA2 knockout has been shown to abrogate formation of intraortic hematopoietic clusters, it is not clear whether GATA2 is required for HE formation, EHT per se, or if it controls HE specification and EHT in a non-cell autonomous manner. To study the function of GATA2 during hematopoietic development, we engineered a GATA2 knockout H1 human embryonic stem cell (hESC) carrying doxycycline (DOX)-inducible GATA2 transgene. We demonstrated that GATA2 deficiency dramatically impaired definitive and primitive hematopoiesis from hESCs. To define the stage of development affected by GATA2, we treated iG2-/- and iG2+/+ hESCs with DOX in stepwise fashion, and evaluated the stage of development at which GATA2 induction has the most profound effect on hematopoiesis. We found that DOX treatment has the most dramatic effect on production of CD43+ cells and CFC potential when performed on days 3-4 or 4-5 of differentiation. In contrast, DOX treatment on days 0-2 suppressed differentiation, while treatment on days 5-6 has little to no effect. Since formation of HE and EHT in our system occurs during days 4-5 of differentiation, i.e. when we see the most dramatic effect of DOX treatment, we concluded that GATA2 may be important for HE formation or EHT. Flow cytometric analysis revealed that formation of VE-cadherin+CD43-CD73- HE, including DLL4+ arterial HE subset, and non-HE was not affected in iG2-/- hESCs, or following GATA2 induction with DOX. Next, we isolated iG2-/- HE from day 4 of differentiation and assessed how DOX treatment affected blood formation. In the absence of DOX, iG2-/-HE produced very few blood cells, while DOX treatment restored blood formation from HE. No significant difference was found in proliferation and in apoptosis of hematopoietic progenitors following EHT, regardless of the presence of GATA2, implying the specific effect of GATA2 on EHT.To define molecular pathways which involved in EHT impairment in iG2-/- cells, we performed RNAseq analysis of HE in DOX- and DOX+ conditions. Gene set enrichment analysis (GSEA) detected significant upregulation of sets of genes responding to TGFβ stimulation in iG2-/- HE on day 1 of secondary culture without DOX as compared to DOX treated cells, thereby suggesting that upregulation of target genes of TGFβ signaling may contribute to EHT impairment in GATA2-deficient cells. Indeed, treatment of iG2-/- HE with TGFβ-inhibitor significantly increased EHT and CD43+ hematopoietic progenitor production with CFU-G and GM potential. Overall, our studies suggest that GATA2 is dispensable for specification of HE, but is essential for EHT and proper hematopoietic specification. In addition, inhibiting TGFβ signaling partially restored the GATA2-dependent EHT in the absence of GATA2. DisclosuresNo relevant conflicts of interest to declare.

Phase 1b Results of a Study to Assess the Efficacy and Safety of Vismodegib in Combination with Ruxolitinib in Patients with Intermediate- or High-Risk Myelofibrosis

Background: The JAK inhibitor (JAKi) ruxolitinib is approved for treatment of myelofibrosis (MF). Treatment for 5 years or more may be necessary to improve or stabilize bone marrow fibrosis, and a fraction of patients do not respond to ruxolitinib. The Hedgehog signaling pathway is involved in primitive and definitive bone marrow hematopoiesis, and maintenance of hematopoietic precursors, suggesting that addition of a Hedgehog pathway inhibitor (HPI) to ruxolitinib might improve responses. In a murine model of MF, a combination of a JAKi and HPI reduced fibrosis in the bone marrow and spleen and decreased splenomegaly. Vismodegib is a HPI approved for treatment of locally advanced and metastatic basal cell carcinoma. The MYLIE study aimed to assess the safety and efficacy of combined ruxolitinib and vismodegib for the treatment of MF. The pharmacokinetics (PK) of vismodegib were characterized in patients with intermediate or high-risk MF.Methods: In this phase 1b study, 10 patients with intermediate- or high-risk MF (per Dynamic International Prognostic Scoring System) received open-label vismodegib (150 mg/day orally) and ruxolitinib (15 or 20 mg orally twice daily, depending on baseline platelet count) for up to 48 weeks, or until withdrawal or discontinuation. PK samples were collected throughout the study. PK sampling was designed to characterize the PK of total and unbound vismodegib in this study for comparison with PK data collected in other patient populations. Efficacy outcomes assessed for phase 1b included spleen response (≥35% reduction in volume by computed tomography or magnetic resonance imaging) per central and investigator assessment, improvement in bone marrow fibrosis per central and investigator assessment (using European consensus grading system), symptom response (≥50% reduction in Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score [MPN-SAF TSS]) and anemia response (per International Working Group for Myeloproliferative Neoplasms Research and Treatment [IWG-MRT] revised response criteria). We report results from the planned analysis of the phase 1b study for efficacy and safety at week 24.Results: Six patients with primary MF, 1 with post-polycythemia vera MF and 3 with post-essential thrombocythemia MF were treated with vismodegib and ruxolitinib for a median of 294.0 days. As of the interim analysis data cutoff (March 29, 2017), 4 patients had completed 48 weeks of study treatment with ruxolitinib and vismodegib, 5 patients were still receiving treatment, and 1 had discontinued treatment because of a vismodegib-related adverse event (AE) (dysgeusia). The most common AEs were muscle spasm (100% of patients), dysgeusia (50%), alopecia (50%), thrombocytopenia (50%), and nausea (40%). All were grade 1/2, except for 2 instances of grade 3 thrombocytopenia in 2 patients. Three patients experienced serious AEs: infection (3 patients), lung infiltration (1 patient), and vomiting (1 patient); none were considered by the investigators to be related to vismodegib. Because of AEs, 7 patients modified or interrupted their ruxolitinib dose, and 4 patients interrupted their vismodegib dose. At week 24, three patients achieved a ≥35% reduction in spleen volume (including the patient who discontinued early because the assessment occurred within the week 24 visit window) by central review (a spleen response occurred in 1 patient per the investigator assessment). According to the central reviews, no patients had improvement in bone marrow fibrosis (1 patient had improvement in bone marrow fibrosis per investigator assessment). Five patients experienced symptom response, and no patients experienced an anemia response. A further central review of bone marrow biopsy specimens from 5 patients at week 48 continued to show no improvement in fibrosis.Conclusions: There was no clear benefit of the addition of vismodegib to ruxolitinib in any of the phase 1b efficacy outcome measures assessed. Given the lack of evidence of a disease-modifying effect with addition of vismodegib to ruxolitinib, further evaluation of this combination will not be pursued.Source of funding: F. Hoffmann-La Roche DisclosuresKoschmieder:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Roche: Other: Clinical Trial participation; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Verstovsek:Incyte: Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Blueprint Medicines Corp: Research Funding; Galena BioPharma: Research Funding; Celgene: Research Funding; NS Pharma: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Lilly Oncology: Research Funding; CTI BioPharma Corp: Research Funding; Astrazeneca: Research Funding; Promedior: Research Funding; Pfizer: Research Funding; Astrazeneca: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Celgene: Research Funding; CTI BioPharma Corp: Research Funding; Galena BioPharma: Research Funding; Roche: Research Funding; Pfizer: Research Funding; Lilly Oncology: Research Funding; Roche: Research Funding. Hooper:Roche Products Ltd: Employment. Tandon:F. Hoffmann-La Roche: Employment. Dimier:F. Hoffmann-La Roche: Employment, Other: Stock.

NOTCH Activation at the Definitive Mesoderm Stage Facilitates Efficient Generation of T Cells with High Proliferation Potential from Human Pluripotent Stem Cells

Adoptive T cell therapies show promise in the treatment of several types of blood cancers. Developing off-the-shelf T cell products will further advance immunotherapies to the clinic and broaden their application. Human pluripotent stem cells (hPSCs) offer the potential to serve as a versatile and scalable source of T cells for immunotherapies, which could be coupled with genetic engineering technologies to meet specific clinical needs. However, production and expansion of T cells from hPSCs remains inefficient. In order to improve T cell production from hPSCs it is essential to identify cell subsets that are highly enriched in T cell progenitors, and those stages of development at which NOTCH activation induces the most potent T cells. Previously, we have developed both OP9-based and chemically defined systems for hematopoietic differentiation from iPSCs (Vodyanik et al., 2006 and Uenishi et. al, 2014). In these differentiation systems, hPSCs undergo stepwise progression towards APLNR+PDGFRa+ mesoderm with hemangioblast colony forming cells (HB-CFCs) that reflect primitive hematopoiesis (day 3 of differentiation), KDRbrightPDGFRalow/- hematovascular mesodermal progenitors (HVMP) with definitive hematopoietic potential, VE-cadherin (VEC)+CD43-CD73- HE with definitive hematopoietic potential (day 4-5 of differentiation) and CD43+ hematopoietic progenitors, including CD235+CD41+ erythromegakaryocytic progenitors (E-MkP) and CD235a/41a-CD45+/- multipotent hematopoietic progenitors (MHP) that have lin-CD34+CD90+CD38-CD45RA- hematopoietic stem progenitor cells (HSPC) phenotype (days 6-8 of differentiation). To assess the stage at which NOTCH activation induces the most potent T cells, we isolated the aforementioned blood forming populations and cultured them in T cell conditions on OP9-DLL4. We found that Day 3 APLNR+PDGFRa+ primitive posterior mesodermal cells did not produce T cells, while all downstream subsets except CD235a+CD41a+CD45- cells do produce T cells when cultured on OP9-DLL4. As determined by limiting dilution assay, the highest frequency of T cell precursors was detected from day 4 HVMP (1 in 14 HVMP). The frequency of T cell precursors in day 5 HE and day 8 HPs was 1 in 16 HEs and 1 in 20 MHPs, respectively. In addition, we found that T cells generated from HVMPs have the capacity to proliferate for 8 weeks, in comparison to HEs and MHPs subsets, which could only be expanded for 4-5 weeks. T cell differentiation from hPSCs proceeded through a CD5+CD7+ progenitor stage that eventually transitions into CD8+CD4+ double positive cells (~90%), CD3+TCRa/b+ and CD3+TCRg/d+ cells. To confirm T cell development, we analyzed the genomic DNA of the hematopoietic cells from OP9-DLL4 cultures for the presence of T cell receptor (TCR) rearrangements. This analysis demonstrated the presence of multiple PCR products of random V-J and D-J rearrangements at the β locus and V-J rearrangements at the γ locus, indicative of a polyclonal T lineage repertoire. In vitro generated T-cells were functionally active and proliferated upon stimulation with PMA and IL-2. Upon activation, the cells express CD25+CD69+ (~73%) markers, cytokines (IFN-γ ~87%, TNFa~22%, IL2 ~34.5%) and cytolytic proteins (Perforin~37%). We also demonstrated that CD5+CD7+ T cell progenitors can be genetically modified to express CD19 CARs and eventually differentiate into CAR T cells with significant cytotoxic effect on Raji cells. Overall, our studies may aid in establishing protocols for the efficient off-the shelf production and expansion of PSC-derived CAR T cells for treating hematologic malignancies. DisclosuresNo relevant conflicts of interest to declare.

Gata2-L359V impairs primitive and definitive hematopoiesis and blocks cell differentiation in murine chronic myelogenous leukemia model

GATA2, a key transcription factor in hematopoiesis, is frequently mutated in hematopoietic malignancies. How the GATA2 mutants contribute to hematopoiesis and malignant transformation remains largely unexplored. Here, we report that Gata2-L359V mutation impeded hematopoietic differentiation in murine embryonic and adult hematopoiesis and blocked murine chronic myeloid leukemia (CML) cell differentiation. We established a Gata2-L359V knockin mouse model in which the homozygous Gata2-L359V mutation caused major defects in primitive erythropoiesis with an accumulation of erythroid precursors and severe anemia, leading to embryonic lethality around E11.5. During adult life, the Gata2-L359V heterozygous mice exhibited a notable decrease in bone marrow (BM) recovery under stress induction with cytotoxic drug 5-fluorouracil. Using RNA sequencing, it was revealed that homozygous Gata2-L359V suppressed genes related to embryonic hematopoiesis in yolk sac, while heterozygous Gata2-L359V dysregulated genes related to cell cycle and proliferation in BM Lin-Sca1+c-kit+ cells. Furthermore, through chromatin immunoprecipitation sequencing and transactivation experiments, we found that this mutation enhanced the DNA-binding capacity and transcriptional activities of Gata2, which was likely associated with the altered expression of some essential genes during embryonic and adult hematopoiesis. In mice model harboring BCR/ABL, single-cell RNA-sequencing demonstrated that Gata2-L359V induced additional gene expression profile abnormalities and partially affected cell differentiation at the early stage of myelomonocytic lineage, evidenced by the increase of granulocyte–monocyte progenitors and monocytosis. Taken together, our study unveiled that Gata2-L359V mutation induces defective hematopoietic development and blocks the differentiation of CML cells.

Mutation of Gemin5 Causes Defective Hematopoietic Stem/Progenitor Cells Proliferation in Zebrafish Embryonic Hematopoiesis.

Fate determination and expansion of Hematopoietic Stem and Progenitor Cells (HSPCs) is tightly regulated on both transcriptional and post-transcriptional level. Although transcriptional regulation of HSPCs have achieved a lot of advances, its post-transcriptional regulation remains largely underexplored. The small size and high fecundity of zebrafish makes it extraordinarily suitable to explore novel genes playing key roles in definitive hematopoiesis by large-scale forward genetics screening. Here, we reported a novel zebrafish mutant line gemin5cas008 with a point mutation in gemin5 gene obtained by ENU mutagenesis and genetic screening, causing an earlier stop codon next to the fifth WD repeat. Gemin5 is an RNA-binding protein with multifunction in post-transcriptional regulation, such as regulating the biogenesis of snRNPs, alternative splicing, stress response, and translation control. The mutants displayed specific deficiency in definitive hematopoiesis without obvious defects during primitive hematopoiesis. Further analysis showed the impaired definitive hematopoiesis was due to defective proliferation of HSPCs. Overall, our results indicate that Gemin5 performs an essential role in regulating HSPCs proliferation.

The transcriptional regulator GON4L is required for viability and hematopoiesis in mice

The Gon4l gene encodes a putative transcriptional regulator implicated in the control of both cell differentiation and proliferation. Previously, we described a mutant mouse strain called Justy in which splicing of pre-mRNA generated from Gon4l is disrupted. This defect severely reduces, but does not abolish, GON4L protein expression and blocks the formation of early B-lineage progenitors, suggesting Gon4l is required for B-cell development in vertebrates. Yet, mutations that disable Gon4l in zebrafish impair several facets of embryogenesis that include the initiation of primitive hematopoiesis, arguing this gene is needed for multiple vertebrate developmental pathways. To better understand the importance of Gon4l in mammals, we created mice carrying an engineered version of Gon4l that can be completely inactivated by Cre-mediated recombination. Breeding mice heterozygous for the inactivated Gon4l allele failed to yield any homozygous-null offspring, indicating Gon4l is an essential gene in mammals. Consistent with this finding, as well previously published results, cell culture studies revealed that loss of Gon4l blocks cell proliferation and compromises viability, suggesting a fundamental role in the control of cell division and survival. Studies using mixed bone marrow chimeras confirmed Gon4l is required for B-cell development but also found it is needed to maintain definitive hematopoietic stem/progenitor cells that are the source of all hematopoietic cell lineages. Our findings reveal Gon4l is an essential gene in mammals that is required to form the entire hematopoietic system.

Heightened activation of embryonic megakaryocytes causes aneurysms in the developing brain of mice lacking podoplanin

AbstractDuring early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1–induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.

The versican-hyaluronan complex provides an essential extracellular matrix niche for Flk1+ hematoendothelial progenitors

Little is known about extracellular matrix (ECM) contributions to formation of the earliest cell lineages in the embryo. Here, we show that the proteoglycan versican and glycosaminoglycan hyaluronan are associated with emerging Flk1+ hematoendothelial progenitors at gastrulation. The mouse versican mutant Vcanhdf lacks yolk sac vasculature, with attenuated yolk sac hematopoiesis. CRISPR/Cas9-mediated Vcan inactivation in mouse embryonic stem cells reduced vascular endothelial and hematopoietic differentiation within embryoid bodies, which generated fewer blood colonies, and had an impaired angiogenic response to VEGF165. Hyaluronan was severely depleted in Vcanhdf embryos, with corresponding upregulation of the hyaluronan-depolymerase TMEM2. Conversely, hyaluronan-deficient mouse embryos also had vasculogenic suppression but with increased versican proteolysis. VEGF165 and Indian hedgehog, crucial vasculogenic factors, utilized the versican-hyaluronan matrix, specifically versican chondroitin sulfate chains, for binding. Versican-hyaluronan ECM is thus an obligate requirement for vasculogenesis and primitive hematopoiesis, providing a vasculogenic factor-enriching microniche for Flk1+ progenitors from their origin at gastrulation.

Tissue-Resident Macrophage Development and Function.

Tissue-resident macrophages have been associated with important and diverse biological processes such as native immunity, tissue homeostasis and angiogenesis during development and postnatally. Thus, it is critical to understand the origins and functions of tissue-resident macrophages, as well as mechanisms underlying their regulation. It is now well accepted that murine macrophages are produced during three consecutive waves of hematopoietic development. The first wave of macrophage formation takes place during primitive hematopoiesis, which occurs in the yolk sac, and gives rise to primitive erythroid, megakaryocyte and macrophage progenitors. These “primitive” macrophage progenitors ultimately give rise to microglia in the adult brain. The second wave, which also occurs in the yolk sac, generates multipotent erythro-myeloid progenitors (EMP), which give rise to tissue-resident macrophages. Tissue-resident macrophages derived from EMP reside in diverse niches of different tissues except the brain, and demonstrate tissue-specific functions therein. The third wave of macrophages derives from hematopoietic stem cells (HSC) that are formed in the aorta-gonad-mesonephros (AGM) region of the embryo and migrate to, and colonize, the fetal liver. These HSC-derived macrophages are a long-lived pool that will last throughout adulthood. In this review, we discuss the developmental origins of tissue-resident macrophages, their molecular regulation in specific tissues, and their impact on embryonic development and postnatal homeostasis.

Can Ultrasound Analysis of the Yolk Sac be a Predictor of Pregnancy Outcome?

The Yolk sac is the first source of transfer between the mother and the embryo, with a nutritional and gas exchange function, vital for the development of the embryo, to which we can add primitive hematopoiesis, the production of stem cells and germ cells. Although normal-term pregnancies with abnormal aspects of the yolk sac have been described, the smaller or larger size of the yolk sac is associated with pregnancy loss. Our study aimed to determine whether the yolk sac size change, determined by measuring diameter (2D ultrasonography) or volume (3D ultrasonography), is independently associated with adverse pregnancy outcomes. The results of the study did not show a statistical significance between 2D and 3D measurements with adverse pregnancy outcomes, noting only an abrupt increase in the diameter and volume of the yolk sac preceding pregnancy loss. However, the evaluation of the yolk sac remains an important element in the ultrasound evaluation of pregnancy in the first trimester

Cohesin Components Stag1 and Stag2 Differentially Influence Haematopoietic Mesoderm Development in Zebrafish Embryos

Cohesin is a multiprotein complex made up of core subunits Smc1, Smc3, and Rad21, and either Stag1 or Stag2. Normal haematopoietic development relies on crucial functions of cohesin in cell division and regulation of gene expression via three-dimensional chromatin organization. Cohesin subunit STAG2 is frequently mutated in myeloid malignancies, but the individual contributions of Stag variants to haematopoiesis or malignancy are not fully understood. Zebrafish have four Stag paralogues (Stag1a, Stag1b, Stag2a, and Stag2b), allowing detailed genetic dissection of the contribution of Stag1-cohesin and Stag2-cohesin to development. Here we characterize for the first time the expression patterns and functions of zebrafish stag genes during embryogenesis. Using loss-of-function CRISPR-Cas9 zebrafish mutants, we show that stag1a and stag2b contribute to primitive embryonic haematopoiesis. Both stag1a and stag2b mutants present with erythropenia by 24 h post-fertilization. Homozygous loss of either paralogue alters the number of haematopoietic/vascular progenitors in the lateral plate mesoderm. The lateral plate mesoderm zone of scl-positive cells is expanded in stag1a mutants with concomitant loss of kidney progenitors, and the number of spi1-positive cells are increased, consistent with skewing toward primitive myelopoiesis. In contrast, stag2b mutants have reduced haematopoietic/vascular mesoderm and downregulation of primitive erythropoiesis. Our results suggest that Stag1 and Stag2 proteins cooperate to balance the production of primitive haematopoietic/vascular progenitors from mesoderm.

Characterization and generation of human definitive multipotent hematopoietic stem/progenitor cells

Definitive hematopoiesis generates hematopoietic stem/progenitor cells (HSPCs) that give rise to all mature blood and immune cells, but remains poorly defined in human. Here, we resolve human hematopoietic populations at the earliest hematopoiesis stage by single-cell RNA-seq. We characterize the distinct molecular profiling between early primitive and definitive hematopoiesis in both human embryonic stem cell (hESC) differentiation and early embryonic development. We identify CD44 to specifically discriminate definitive hematopoiesis and generate definitive HSPCs from hESCs. The multipotency of hESCs-derived HSPCs for various blood and immune cells is validated by single-cell clonal assay. Strikingly, these hESCs-derived HSPCs give rise to blood and lymphoid lineages in vivo. Lastly, we characterize gene-expression dynamics in definitive and primitive hematopoiesis and reveal an unreported role of ROCK-inhibition in enhancing human definitive hematopoiesis. Our study provides a prospect for understanding human early hematopoiesis and a firm basis for generating blood and immune cells for clinical purposes.

Zrsr2 Deficient Zebrafish Display Hematopoietic Defects and U12-Type Intron Retention in mRNA Processing Genes

ZRSR2 is a small nuclear riboprotein necessary for assembly of the minor spliceosome and subsequent splicing of U12-type introns. It is involved in the recognition of 3' splice sites during the assembly of the spliceosome. Somatic mutations in ZRSR2 gene are present in hematopoietic malignancies, most notably myelodysplastic syndrome (MDS). These mutations are spread throughout the gene, indicating that its function is critical for hematopoiesis. In spite of these clinical associations, the role of ZRSR2 in hematopoiesis has not been well studied. Zebrafish make an excellent animal model for investigating this gene as all of the functional domains in the human protein are conserved with a high level of protein similarity in zebrafish. Additionally, hematopoietic development is well understood in this species. We used CRISPR/Cas9 to generate a zebrafish zrsr2 knockout model with an 11 base pair deletion (zrsr2Δ11) that results in frameshift with premature truncation and loss of all functional domains (p.W167fs*175). The zrsr2Δ11/Δ11 mutants began to appear morphologically different from their siblings by 4 days post fertilization (dpf), showing aberrant development in the mandible and pharyngeal arches. The zrsr2Δ11/Δ11 mutants developed mild to severe edema by 6 dpf and died by 8 dpf. To understand its role in hematopoiesis, we performed o-dianisidine staining and whole mount in situ hybridization (WISH) with lineage-specific markers during embryonic development. Our data showed that zrsr2Δ11/Δ11 embryos exhibit mild anemia by 2 dpf, suggesting that primitive hematopoiesis is defective. WISH showed that zrsr2Δ11/Δ11 mutants have normal early hematopoietic stem cell (HSC) emergence (c-myb) at 36 hours post fertilization. However, these cells are absent through 3 and 5 dpf. At 5 dpf zrsr2Δ11/Δ11 mutants had severely reduced expression of hematopoietic markers for erythroid (hbae1), lymphoid (rag1), and myeloid (mpo) lineages. This verifies that knockout of zrsr2 in zebrafish disrupted normal hematopoiesis and leads to cytopenias - emulating the symptoms of MDS. We then investigated the transcriptional and splicing changes underlying these phenotypes using RNA sequencing on zrsr2Δ11/Δ11 mutant embryos at 3dpf. We found a total of 285 intron retention events in zrsr2Δ11/Δ11 mutants as compared to the control embryos, with about 76% of those events occurring within U12-type introns. Genes with retained introns were associated with mRNA decay and destabilization. We also found that genes related to cell cycle arrest were upregulated in zrsr2Δ11/Δ11 mutants . These results indicate that aberrant mRNA splicing and cell cycle deregulation contribute to arrested hematopoiesis when zrsr2 is knocked out in zebrafish. Disclosures No relevant conflicts of interest to declare.

Utilizing CRISPR-CAS9 Gene Editing Technology in Human Pluripotent Stem Cells to Study Platelet Integrin αIIbβ3 Function

Integrins are a family of cell surface receptors that transmit signals bidirectionally across plasma membranes. Cellular agonists induce 'inside-out' signals that cause ligand binding to integrins; in turn, this causes integrin-mediated 'outside-in' signals that control cellular functions such as adhesion and migration. The platelet integrin αIIbβ3 has served as a paradigm for deciphering these bidirectional signaling mechanisms. Moreover, site-directed mutagenesis has been essential for dissecting integrin structure-function relationships. For platelet integrins such as αIIbβ3, the structure-function consequences of the site-directed mutations have previously been studied in epithelial cell lines, such as Chinese hamster ovary (CHO) cells. Only recently has technology advanced sufficiently to enable study of platelet integrins in their normal cellular environment, the platelet-producing megakaryocyte. The advent of human induced pluripotent stem cells (iPSC), coupled with the ability to gene edit these cells using the CRISPR-CAS9 technology provides a way to study αIIbβ3 mutants in megakaryocytes. Following platelet stimulation, αIIbβ3 rapidly undergoes a global rearrangement in which a clasp composed of its extracellular stalk, transmembrane, and membrane-proximal cytoplasmic domains is disrupted causing the αIIbβ3 headpiece to open exposing a ligand binding site. Using computational methods, we predicted mutations that would destabilize the stalk domain, thereby causing αIIbβ3 activation. These predictions were confirmed by showing that the mutations were indeed able to cause constitutive αIIbβ3 activation in CHO cells (Donald et al, J. Biol. Chem. 2010; Tan et al, Biochemistry 2019). To translate these findings to human cells and compare the functional consequences of the mutations when endogenously expressed in iPSC-derived megakaryocytes, we chose a highly activating V760A missense mutation located in the stalk region of the αIIb subunit. Using an established control iPSC line designated CHOPWT14, both heterozygous and homozygous V760A missense mutations were created in the ITGA2B gene encoding αIIb using our CRISPR-CAS9 protocol (Maguire et al. Current Protocols in Stem Cell 2019). The generation of these mutant cell lines from a genetically identical control cell line eliminates the heterogeneity and variability in downstream applications that was observed when using lines of different genetic backgrounds. To ensure genetic identity of the parent and mutant lines, copy number variation or CNV analyses were performed and shown to be identical in all lines. To study ligand binding of αIIbβ3 expressing the V760A mutation in iPSC-derived megakaryocytes, the isogenic control and mutant lines were differentiated to blood using our serum- and stromal-free directed differentiation protocol (Mills et al. Methods in Mol. Biol. 2014). Briefly, the differentiation of human iPSCs to blood follows primitive hematopoiesis which recapitulates the developmental stages observed in the embryo of primitive streak formation, mesoderm specification, and hematopoietic development, including megakaryocytes. To measure conformational change in αIIbβ3, the activation-dependent monoclonal antibody PAC-1 was used as a surrogate for fibrinogen. By analyzing CD41+CD42b+ megakaryocytes by flow cytometry, PAC-1 was shown to bind constitutively to 23.4% and 26.0% of the megakaryocytes expressing heterozygous and homozygous V760A mutations, respectively, compared to 9.04% of control megakaryocytes. The specificity of PAC-1 binding was confirmed by a reduction in binding to <1% following pretreatment with EDTA. In addition, thrombin stimulation increased PAC-1 binding to >65% in all lines, indicating normal overall αIIbβ3 function. Essentially identical results were obtained when FITC-fibrinogen was used instead of PAC-1. These data show that 1) structure-function studies of computationally identified mutations confirmed in CHO cells can be analyzed using human iPSC-derived megakaryocytes, 2) mutations shown to be highly active in CHO cells appear to be constrained or less constitutively active in human megakaryocytes, and 3) more in-depth analyses of platelet integrin structure-function relationships will be possible using human megakaryocytes. Disclosures No relevant conflicts of interest to declare.

Manipulation of Tropomysin 1 in iPSCs to Enhance in Vitro Blood Cell Production

Donor-derived blood transfusions are critical to our healthcare system, but do not fully meet the needs of patients with multiple alloantibodies, rare blood types, or HLA-sensitization. These needs have fueled the concept of ex vivo blood cell production, which might address issues related to demographic aging, infectious outbreaks transmitted by transfusions, and rare blood types. Blood cells produced in vitro could be used for transfusions, and could also be used as blood bank testing reagents (Coleman et al, Transfusion 2019). One major challenge in deploying this system is efficiently scaling up cell production. We used machine learning and genome-edited induced pluripotent stem cell (iPSC) models to determine that Tropomyosin 1 (TPM1) normally inhibits in vitro hematopoiesis. TPM1 knockout (TPM1KO) iPSCs produced 2-fold more hematopoietic progenitor cells (HPCs) than controls, thereby increasing production of mature blood cells that were functionally normal (Thom et al, BMC Biol 2020). During human hematopoiesis, HPCs arise from specialized vascular 'hemogenic endothelial' cells (HE) with distinct surface markers that can be used for identification and isolation. To define molecular mechanisms by which TPM1 regulates in vitro primitive hematopoiesis, we performed RNA sequencing analysis on sorted KDR+CD31+ endothelial cells and CD43+ HPCs from TPM1KO and control cultures. TPM1KO endothelial cells and HPCs had altered expression of genes and pathways known to regulate HE biology, including cell adhesion, integrin expression, and integrin-mediated signaling (p<0.05). 'Anoikis' is an apoptosis-like programmed cell death that occurs after extracellular matrix detachment. This process may limit nascent non-adherent HPC production in vitro, but has not been previously studied. TPM1KO cells showed increased expression of N-cadherin and RAP1-activating genes; increased N-cadherin and activated RAP1 limit anoikis in other biological contexts. In sum, these results suggested that TPM1KO cultures increased HE production and/or survival. To analyze HE production at the single cell level, we sorted KDR+CD31+CD43- endothelial cells and plated them in limiting dilution. We cultured sorted cells in hematopoietic cytokines for 7 days, and analyzed the number of wells in which CD43+ HPCs arose from sorted TPM1KO and control cells. Using limiting dilution analysis (Hu & Smyth,J. Immunol. Methods 2009), we found that TPM1KO cultures produced 2-fold more HE than controls. These results show that TPM1KO enhances in vitro hematopoiesis by increasing HE and subsequent HPC production, perhaps by limiting anoikis in nascent HPCs. TPM1-mediated regulation at the HE stage represents a novel mechanism that may be genetically or pharmacologically exploited to augment in vitro hematopoiesis. These findings will help boost in vitro HPC and blood cell production to clinically relevant scales, supporting efforts to produce blood cells for direct transfusion and/or to be used as clinical screening reagents. Disclosures No relevant conflicts of interest to declare.

Notch signaling and the emergence of hematopoietic stem cells.

The hematopoietic stem cell (HSC) is able to give rise to all blood cell lineages in vertebrates. HSCs are generated in the early embryo after two precedent waves of primitive hematopoiesis. Canonical Notch signaling is at the center of the complex mechanism that controls the development of the definitive HSC. The successful in vitro generation of hematopoietic cells from pluripotent stem cells with the capacity for multilineage hematopoietic reconstitution after transplantation requires the recapitulation of the most important process that takes place in the hemogenic endothelium during definitive hematopoiesis, that is the endothelial-to-hematopoietic transition (EHT). To meet this challenge, it is necessary to thoroughly understand the molecular mechanisms that modulate Notch signaling during the HSC differentiation process considering different temporal and spatial dimensions. In recent years, there have been important advances in this field. Here, we review relevant contributions describing different genes, factors, environmental cues, and signaling cascades that regulate the EHT through Notch interactions at multiple levels. The evolutionary conservation of the hematopoietic program has made possible the use of diverse model systems. We describe the contributions of the zebrafish model and the most relevant ones from transgenic mouse studies and from in vitro differentiated pluripotent cells.

Common Sources of Inflammation and Their Impact on Hematopoietic Stem Cell Biology.

Purpose of ReviewInflammatory signals have emerged as critical regulators of hematopoietic stem cell (HSC) function. Specifically, HSCs are highly responsive to acute changes in systemic inflammation and this influences not only their division rate but also their lineage fate. Identifying how inflammation regulates HSCs and shapes the blood system is crucial to understanding the mechanisms underpinning these processes, as well as potential links between them.Recent FindingsA widening array of physiologic and pathologic processes involving heightened inflammation are now recognized to critically affect HSC biology and blood lineage production. Conditions documented to affect HSC function include not only acute and chronic infections but also autoinflammatory conditions, irradiation injury, and physiologic states such as aging and obesity.SummaryRecognizing the contexts during which inflammation affects primitive hematopoiesis is essential to improving our understanding of HSC biology and informing new therapeutic interventions against maladaptive hematopoiesis that occurs during inflammatory diseases, infections, and cancer-related disorders.

3098 – THE ROLE OF GATA2 IN HSC GENERATION THROUGH ENDOTHELIAL-TO-HEMATOPOIETIC TRANSITION

GATA2 is the most frequently mutated gene in childhood AML. These patients have germline mutations in one allele of GATA2 and have a high predisposition for MDS/AML. Also, in mouse, Gata2 is required for embryonic HSC generation and survival and Gata2 haploinsufficiency results in a severe reduction in the formation of HSCs. The first HSCs are formed at embryonic day (E)10.5 through a process called endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial cells of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region. As all patients suffering from GATA2 haploinsufficiency syndromes have innate GATA2 mutations we want to understand how Gata2 haploinsufficiency affects embryonic HSC generation. Therefore, we sorted phenotypic HSPCs (CD31+cKit+ cells) from WT and Gata2+/- E11 AGMs and performed transcriptome analysis. Surprisingly, we found that the hematopoietic transcriptional program is not abrogated in Gata2+/- mouse E11 CD31+cKit+ cells. However, Gata2+/- HSPCs still express the endothelial program indicating that the hematopoietic cells are stuck in the endothelium and cannot complete EHT. We found that Gfi1b is downregulated in Gata2+/- HSPCs. As Gfi1b is known to be required for downregulation of the endothelial program for EHT during primitive hematopoiesis, we hypothesize that Gata2 directly regulates Gfi1b and that this regulation is required for normal EHT. We are currently studying if a specific HSPC subtype can overcome Gata2 haploinsufficiency and will still undergo EHT as this may have implications for the hematopoietic cells that eventually populate the hematopoietic tissues.

1008 – INTERFERON GAMMA-MEDIATED REGULATION OF HSCS: MECHANISMS AND ROLE IN CLONAL HEMATOPOIESIS.

Inflammatory conditions such as chronic infection or autoimmune disease are frequently associated with impaired hematopoiesis. Chronic interferon gamma (IFNγ)-mediated inflammatory signaling damages hematopoietic stem cells (HSC) by disrupting quiescence, promoting excessive terminal differentiation, and increasing the propensity for secondary stress-induced apoptosis. In recent studies, we examined the mechanisms underlying IFNγ-mediated HSC activation. Using intravital three-dimensional microscopy, we found that IFNγ disrupts the normally close interaction between HSCs and CXCL12-abundant reticular (CAR) cells in the HSC niche. IFNγ-dependent relocalization and HSC activation are dependent on the cell surface protein BST2, which is induced by IFNγ. HSCs from mice lacking BST2 were more quiescent and resistant to depletion upon chronic infection. Thus, BST2 is a key regulator of HSC quiescence that promotes inflammation-induced niche relocalization and activation. Given that prolonged IFNγ signaling can lead to the depletion of HSCs, we examined the impact of IFNγ-mediated HSC responses on clonal hematopoiesis (CH), with particular attention to loss of function (LOF) mutations in <i>Dnmt3a</i>, the most commonly mutated gene in CH. We used a murine model to investigate the prediction that infection provides selective pressure favoring expansion of <i>Dnmt3a</i>-knock out HSCs. We created <i>Dnmt3a</i> mosaic mice by transplanting a mixed population of Dnmt3a-KO and WT HSCs into WT mice and observed substantial expansion of Dnmt3a-KO HSCs during chronic mycobacterial infection. Transcriptional profiling and functional studies indicate reduced differentiation and reduced secondary stress-induced apoptosis account for <i>Dnmt3a</i> KO clonal expansion during infection. Whole genome bisulfite sequencing reveals possible mechanisms for these effects. Thus, we demonstrate that IFNg signaling during chronic infection can drive DNMT3A-mutant CH. Our future work will continue to elucidate the avenues by which infections and inflammation alter primitive hematopoiesis.