Primitive Blood Cells Research Articles

Sars-Cov-2 Spike Protein Induces Cellular Changes in Primitive and Mature Hematopoietic Cells

The devastating effects of SARS-CoV-2 infection (which causes COVID-19) highlight the need for a deeper understanding of the virus, host response, and the tissues affected by infection, including the hematopoietic stem cells (HSC), hematopoietic progenitors (HPC), and more mature blood cells including immune cells. While other coronavirus infections have been associated with hematopoietic or immune cell complications (Chu et al., J Infect Dis 2016), the effects of SARS-CoV-2 infection on HSC/HPC and mature immune cells are not yet understood. We performed studies to examine the potential susceptibility of HSC/HPC subpopulations and immune cells to infection and functional studies to explore if exposure to SARS-CoV-2 proteins impacts these cells. The most well-studied receptor for SARS-CoV-2 is ACE2 (Hoffmann et al., Cell 2020), a cell surface protein to which SARS-CoV-2 spike protein (S protein) binds to facilitate viral entry to host cells. We explored expression of ACE2 in primitive and mature blood cells. We demonstrated using RT-qPCR as well as western blotting that ACE2 is expressed at both the mRNA and protein levels in pooled low-density peripheral blood cells (PB) from healthy donors (n=5); pooled CD34+ cells from cord blood (CB) enriched for HSC/HPC (n=7); lineage+ (lin+) low density CB cells (n=7), and high density polymorphonuclear leukocytes (PMN) from CB (n=3). We then determined ACE2 expression on specific subpopulations of HSC/HPC and immune cells. Flow cytometry analysis (FACS) demonstrated that ACE2 is expressed on the cell surface of small subpopulations of PB immune cells, including 1-2% of T-cells, 2-4% of B-cells, and <1% of NK cells and monocytes. However, larger populations of HSC/HPC exhibited ACE2 cell surface expression. The percent ACE2+ HSC, multipotent progenitor cells (MPP), and multipotent lymphoid progenitor cells (MLP) were respectively 15-60%, 5-50%, and 5-15% of cells (n=5). We next determined if exposure to viral S protein affects these cell populations. We first tested if SARS-CoV-2 S protein induces responses in HSC/HPC. We isolated CD34+ cells from low density CB and expanded them in the presence of serum and growth factors (100ng/mL TPO, FLT3L, and SCF) in the presence or absence of recombinant full-length S protein. After 7 days expansion, cells were analyzed for immunophenotype using FACS or were plated in semi-solid methylcellulose in an HPC colony forming unit (CFU) assay and grown for 12 days before scoring for functional CFU-Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) and CFU-Granulocyte/Macrophage (CFU-GM) progenitor cells. CB CD34+ cells exhibit significantly less expansion when exposed to SARS-CoV-2 S protein, showing 33% less expansion of CD34+ cells. The most significant delay in expansion of phenotyped cells is evident in granulocyte-monocyte progenitors (GMP, 38% less expansion), with a more modest 15-30% decrease in expansion for common myeloid progenitors/megakaryocyte-erythroid progenitors (CMP/MEP), HSC, MPP, and MLP (n=4). S protein lessens functional HPC colony forming cell expansion compared to control, with 12-fold and 2.1-fold expansions compared to 30.8-fold and 6-fold expansions respectively for CFU-GM and CFU-GEMM. These data indicate that exposure to SARS-CoV-2 S protein impacts hematopoiesis and myeloid differentiation in vitro. We next tested effects of S protein exposure on PB by isolating low density blood cells from healthy donors and culturing them ex vivo for different time periods in the presence or absence of recombinant full-length S protein. After 2hrs incubation, we observed by FACS a modest but consistent 1.2-fold increase in CD14 positive monocytes in cells exposed to S protein compared vehicle control (n=4). After 18hrs incubation with the S protein, the PB monocytes exhibited consistent changes in morphology, becoming smaller and more granular, determined by changes in forward scatter and side scatter by FACS (n=5). This suggests that exposure to SARS-CoV-2 protein may have a physiologic impact on PB cells ex vivo, despite their low levels of ACE2 expression. Our data thus has important implications for hematopoiesis and immune response in COVID-19 patients. It demonstrates the need to determine whether CB potentially exposed to SARS-CoV-2 should be banked, as the effects we observed may affect the efficacy of CB transplantations.DisclosuresNo relevant conflicts of interest to declare.

MYB bi-allelic targeting abrogates primitive clonogenic progenitors while the emergence of primitive blood cells is not affected.

MYB is a key regulator of definitive hematopoiesis and it is dispensable for the development of primitive hematopoietic cells in vertebrates. In order to delineate definitive versus primitive hematopoiesis during differentiation of human embryonic stem cells, we have introduced reporters into the MYB locus and inactivated the gene by bi-allelic targeting. In order to recapitulate the early developmental events more adequately, mutant and wild-type human embryonic stem cell lines were differentiated in defined culture conditions without the addition of hematopoietic cytokines. The differentiation of the reporter cell lines demonstrated that MYB is specifically expressed throughout emerging hematopoietic cell populations. Here we show that the disruption of the MYB gene leads to severe defects in the development and proliferation of primitive hematopoietic progenitors while the emergence of primitive blood cells is not affected. We also provide evidence that MYB is essential for neutrophil and T-cell development and the upregulation of innate immunity genes during hematopoietic differentiation. Our results suggest that the endothelial origin of primitive blood cells is direct and does not include the intermediate step of primitive hematopoietic progenitors.

CBFA2T3-GLIS2 induces Malignant Transformation of Primitive CD34+ Cord Blood Cells and Recapitulates Infant Megakaryocytic Leukemia

CBFA2T3-GLIS2 fusions are uniquely expressed in infants with AML and its presence is associated with refractory disease with high induction failure and nearly uniform fatality. Recent studies into varying features of this refractory leukemia have defined a unique immunophenotype that is referred to as the RAM phenotype with exceptionally high CD56 expression. Further, deep sequencing of patients with this fusion has failed to identify additional recurrent sequence or structural variants, suggesting that the presence of this fusion might be sufficient for malignant transformation. Given its presence uniquely in infants and lack of cooperating events, the premise of malignant transformation by a single, highly penetrant event might be justified. Despite discovery efforts to define genes, pathways, and networks that are dysregulated in this AML subset, lack of appropriate model systems has limited pre-clinical evaluation of potential therapies. Prior efforts in generating cell lines and xenograft models have met with limited success. We inquired whether primitive CD34+ cord blood cells might provide the appropriate developmental milieu to study fusion-induced transformation. To investigate this possibility, we transduced human CD34+ cord blood cells with a lentivirus encoding CBFA2T3-GLIS2 fusion transcript and GFP. Transduced and mock-transduced control cells were cultured in bulk in a myeloid promoting condition (IMDM with 15% FBS and 10 ng/mL of each growth factor SCF, TPO, FLT3L, IL3, and IL6). The transduction efficiency was approximately 9% at MOI of 50 and expression of fusion transcript was confirmed by RT-PCR. Growth kinetics and surface expressions were assessed by flow cytometry. Evaluation of the growth properties showed that CBFA2T3-GLIS2 transduced cells exhibited enhanced proliferation, with a doubling time (DT) of 93 hours after 5 weeks, whereas DT for mock-transduced control cells was 124 hours (Figure 1A). Further, CBFA2T3-GLIS2 transduced cells had significant expansion of the GFP+ cells, an increase from 9% at the initiation of cultures to 95% by week 8 (Figure 1B). After 12 weeks in culture, fusion-positive cells continue their rapid expansion. Surface CD34 expression was lost after the first week of culture for both CBFA2T3-GLIS2 and mock-transduced cells (Figure 1C), indicative of rapid differentiation in myeloid promoting condition. Morphological evaluation of cells expressing CBFA2T3-GLIS2 reveal a homogenous population with open chromatin, occasional multinucleated cells, prominent nucleoli, and abundant focally basophilic and vacuolated cytoplasm with cytoplasmic blebs (Figure 1D) which are morphologic features suggestive of megakaryocytic differentiation. Megakaryocytic lineage of the fusion transduced cells was confirmed by demonstration of high expression of CD41 and CD61 on the surface of transduced cells (Figure 1E). We further evaluated immunophenotypic alterations as a result of CBFA2T3-GLIS2 fusions, by comparing the surface expression of 16 different markers including CD11b, CD33, CD34, CD36, CD38, CD56, CD64, CD117, CD123, and HLA-DR between CBFA2T3-GLIS2 and mock transduced cells. Mock-transduced cells showed expression of antigens associated with monocytic differentiation (CD36, CD64, and CD11b) (Figure 1F) whereas the CBFA2T3-GLIS2 transduced cells were consistent with more immature cells expressing CD117 and lacking CD11b, CD36 and CD64 (Figure 1G). Of significance, fusion-transduced cells had remarkably high CD56 expression (mean linear MFI of 43 and 3556 for the mock-transduced and transduced cells respectively), similar to that observed in patients with fusion-positive RAM phenotype (average MFI=2040) (Figure 1H), suggesting that CD56 expression is causally linked to the expression of the fusion transcript. This study demonstrates that the CBFA2T3-GLIS2 fusion is sufficient for malignant transformation and recapitulates megakaryocytic AML that is seen in infants. This further demonstrates that engineered primitive cord blood cells can be utilized for generation of appropriate cell lines that can be used for therapeutic development. Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership.

Alpha fetoprotein (AFP) participates in the build up of hematopoietic cells in the early embryonic stage: an abortion case observation

BackgroundAt the 3rd week of human embryo, some cell clumps are formed by the hyperplasia of mesenchymal cells at the germ layer of the yolk sac wall. These cell clumps are known as blood islands. The cells in the center of the blood islands further develop into primitive blood cells, such as hematopoietic stem cells. The blood island in the yolk sac further develops into the extramedullary hematopoietic tissue in 1 week at the 3rd to 4th week.Case presentationA 32-year-old pregnant woman who missed menstruation for 42 days discovered that her pregnancy required an abortion. The tissue collected after the abortion was a piece of gray-yellow and villus-like intrauterine tissue of a size of approximately 4 cm × 3 cm × 1.3 cm. The paraffin section stained with hematoxylin and eosin and observed under the light microscope showed a visible small embryo tissue in the early placental tissue. In the embryonic tissue, a large amount of extramedullary hematopoietic tissue was present, including myeloid, erythroid and megakaryocytic cells. The extramedullary hematopoietic cells were located in the blood vessels or naive liver sinus, were positive for alpha fetoprotein (AFP) and were without lymphocytes. The erythrocytes consisted of a large number of nucleated red blood cells. In addition, a neural tube and cystic structure were found. The final pathological diagnosis was as follows: Early embryonic tissue with a cystic structure formation in the embryo. After medical abortion the pregnant woman recovered well, without complications.ConclusionsOur case illustrates that AFP is an important structural protein of nucleated erythrocytes and myeloid hematopoietic cells, suggesting that it may participate in the build up of nucleated erythrocytes and myeloid hematopoietic cells. Furthermore, our case suggests that nucleated red blood cells can be detected from the 42nd day of pregnancy by a peripheral blood sample from the mother.

The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1

Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury‐positive multipotent cells in the posterior‐proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog‐deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers.

MYB is an Essential Regulator of Primitive Human Hematopoiesis in Pluripotent Stem Cell Differentiation Cultures

MYB is a key regulator of definitive hematopoiesis that plays a critical role in the maintenance and multilineage differentiation of hematopoietic stem cells (HSCs). In vertebrate developmental models, MYB is thought to be dispensable for primitive hematopoiesis. To explore the role of MYB in human hematopoietic development, we have subjected human pluripotent stem cells (hPSCs) to mono- and bi-allelic gene targeting followed by hematopoietic differentiation in defined culture conditions. Here we show that MYB plays a central role in the development of human primitive blood cells. MYB expression was hematopoietic-specific and its induction coincided with emergence of the earliest primitive blood cells. Bi-allelic inactivation of MYB most severely affected the primitive erythroid progenitors of greater proliferative capacity and multilineage hematopoietic progenitors. The initial phase of the hematopoietic differentiation was not affected by the bi-allelic gene deficiency, but maturation of the primitive myeloid cells was found to be MYB-depended. Rescuing MYB expression in MYB-null cells shows that the gene is required for both development and proliferation of primitive clonogenic progenitors. In addition, our findings suggest that human primitive hematopoiesis emerge in several developmental cohorts.

Single-cell analysis identifies a CD33+ subset of human cord blood cells with high regenerative potential.

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.

PLAG1 and USF2 Co-regulate Expression of Musashi-2 in Human Hematopoietic Stem and Progenitor Cells.

SummaryMSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs), enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry.

Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis.

The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however,can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived invitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from hemogenic endothelium? Is the hemangioblast an invitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.

Nucleosome Density ChIP-Seq Identifies Distinct Chromatin Modification Signatures Associated with MNase Accessibility

Nucleosome position, density, and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset ofnormal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis, we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly, extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters, suggesting developmental stage-specific organization of histone methylation states.

Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants

Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models.In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

The Multimerization Domain of Cbfß-SMMHC Is Required for Leukemogenesis

Among acute myeloid leukemia (AML) with cytogenetic abnormalities, core binding factor (CBF) leukemia acounts for 20-30% of adult AML, and 20-30% of pediatric AML. The chromosome 16 inversion (inv(16)), which results in a fusion gene CBFB -MYH11 and an encoded chimeric protein CBFβ-SMMHC (core binding factor β - smooth muscle myosin heavy chain), is observed primarily in AML subtype M4Eo. Using Cbfb-MYH11 knock-in mouse models we previously demonstrated that CBFβ-SMMHC needs its C terminal domains for leukemogenesis (Kamikubo et al, Blood 121:638, 2013). In this study we generated a new CBFB-MYH11 knock-in mouse model to determine the role of the multimerization domain at the C terminus of CBFβ-SMMHC for hematopoietic defects and leukemogenesis.Previous studies have shown that the C-terminal 29-residue assembly competent domain (ACD) is essential for multimerization of SMMHC. Within ACD, clustered point mutations in helices D and E specifically disrupts multimerization of CBFβ-SMMHC without interfering with the repression function of CBFβ-SMMHC (Zhang et al., Oncogene 25:7289, 2006). Therefore, we generated knock-in mice expressing CBFβ-SMMHC with mutated helices D and E (mDE) to study the role of the multimerization domain in vivo.Heterozygous embryos (Cbfb+/mDE) were viable and showed no defects in fetal liver definitive hematopoiesis, while homozygous embryos (CbfbmDE/mDE) showed complete blockage of definitive hematopoiesis, hemorrhage in the central nervous system and midgestation lethality, similar to the phenotype in Cbfb+/MYH11 mice and the Cbfb or Runx1 null mice. This phenotype is also similar to that in the homozygous knockin embryos expressing C-terminally-deleted CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013). The fetal liver of E12.5 CbfbmDE/mDE embryos gave no colonies while the fetal liver of Cbfb+/mDE mice generated similar number of colonies as the WT controls. We further looked at the peripheral blood of E10.5 CbfbmDE/mDE embryos and found that the primitive hematopoiesis was not affected, while E10.5 Cbfb+/MYH11 embryos showed a developmental delay at this stage.Analysis of peripheral blood showed decreased B cell population in young adult Cbfb+/mDE mice, while the myeloid compartment was unchanged. In aged mice (>12 months), however, there was an increase of immature myeloid cells in the peripheral blood. Importantly, there was no leukemia development in the Cbfb+/mDE mice one year after ENU treatment (to induce cooperating mutations), while Cbfb+/MYH11 micedied of leukemia within 2 months of ENU treatment. Notably bone marrow cells in the Cbfb+/mDE and Cbfb+/MYH11 mice expressed their respective fusion proteins at similar levels.Overall our data suggest that the C terminal multimerization domain is required for the defects in primitive and definitive hematopoiesis caused by CBFβ-SMMHC, and the domain is essential for leukemogenesis by CBFβ-SMMHC. Further mechanistic studies of this domain may lead to new drug targets for treating inv(16) leukemia. For this purpose we have performed gene expression profiling with microarray and RNA-seq technologies, comparing gene expression changes in adult bone marrow c-Kit+ cells as well as embryonic primitive blood cells from Cbfb+/mDE and Cbfb+/MYH11 mice. Preliminary analysis indicates that the gene expression profile of the hematopoietic cells from the Cbfb+/mDE mice was much similar to that of Cbfb+/+ than Cbfb+/MYH11 mice. Validation and pathway analysis of those differentially expressed genes are ongoing and the results will be presented at the annual meeting. DisclosuresNo relevant conflicts of interest to declare.

Expression pattern of bcar3, a downstream target of Gata2, and its binding partner, bcar1, during Xenopus development

Primitive hematopoiesis generates red blood cells that deliver oxygen to the developing embryo. Mesodermal cells commit to a primitive blood cell fate during gastrulation and, in order to do so the mesoderm must receive non-cell autonomous signals transmitted from other germ layers. In Xenopus, the transcription factor Gata2 functions in ectodermal cells to generate or transmit the non-cell autonomous signals. Here we have identified Breast Cancer Antiestrogen Resistance 3 (bcar3) as a gene that is induced in ectodermal cells downstream of Gata2. Bcar3 and its binding partner Bcar1 function to transduce integrin signaling, leading to changes in cellular morphology, motility and adhesion. We show that gata2, bcar3 and bcar1 are co-expressed in ventral ectoderm from early gastrula to early tailbud stages. At later stages of development, bcar3 and bcar1 are co-expressed in the spinal cord, notochord, fin mesenchyme and pronephros but each shows additional unique sites of expression. These co-expression and unique expression patterns suggest that Bcar3 and Bcar1 may function together but also independently during Xenopus development.

Neuropilin-2 genomic elements drive cre recombinase expression in primitive blood, vascular and neuronal lineages.

We have established a novel Cre mouse line, using genomic elements encompassing the Nrp2 locus, present within a bacterial artificial chromosome clone. By crossing this Cre driver line to R26R LacZ reporter mice, we have documented the temporal expression and lineage traced tissues in which Cre is expressed. Nrp2-Cre drives expression in primitive blood cells arising from the yolk sac, venous and lymphatic endothelial cells, peripheral sensory ganglia, and the lung bud. This mouse line will provide a new tool to researchers wishing to study the development of various tissues and organs in which this Cre driver is expressed, as well as allow tissue-specific knockout of genes of interest to study protein function. This work also presents the first evidence for expression of Nrp2 protein in a mesodermal progenitor with restricted hematopoietic potential, which will significantly advance the study of primitive erythropoiesis. genesis 53:709-717, 2015. © 2015 Wiley Periodicals, Inc.

Modeling de novo leukemogenesis from human cord blood with MN1 and NUP98HOXD13

AbstractLeukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.

A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells

SummaryDisrupted IKAROS activity is a recurrent feature of some human leukemias, but effects on normal human hematopoietic cells are largely unknown. Here, we used lentivirally mediated expression of a dominant-negative isoform of IKAROS (IK6) to block normal IKAROS activity in primitive human cord blood cells and their progeny. This produced a marked (10-fold) increase in serially transplantable multipotent IK6+ cells as well as increased outputs of normally differentiating B cells and granulocytes in transplanted immunodeficient mice, without producing leukemia. Accompanying T/natural killer (NK) cell outputs were unaltered, and erythroid and platelet production was reduced. Mechanistically, IK6 specifically increased human granulopoietic progenitor sensitivity to two growth factors and activated CREB and its targets (c-FOS and Cyclin B1). In more primitive human cells, IK6 prematurely initiated a B cell transcriptional program without affecting the hematopoietic stem cell-associated gene expression profile. Some of these effects were species specific, thus identifying novel roles of IKAROS in regulating normal human hematopoietic cells.

RhoA Gtpase Regulates Erythroblast Cytokinesis and Enucleation By Dynamic Coordination Of The Microtubule-Actomyosin Machineries For Successful Abscission

RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis. Specifically, it contributes to cytokinesis in sea urchin embryos, Xenopus and HeLa cells by associating with the microtubule ends at the abscission area marking the position for the daughter cell separation and by activating myosin through phosphorylation of the myosin regulatory light chain (MRLC) (Piekny et al, Trends in Cell Biology 2005). Erythroblast enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis (reviewed by Li, Developmental Cell 2013). It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring between reticulocyte and pyrenocyte (Konstantinidis et al, Blood 2012).To define the mechanistic contribution of RhoA signaling in terminal erythroid maturation, we generated mice with erythroid specific RhoA deletion (EpoR-GFPcre-driven). Erythroid-specific deletion of RhoA, confirmed in the CD71-positive cells by PCR and immunoblotting, caused severe anemia, fatal in utero by E15.5. RhoA-deficient peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear with polyploidy of the genetic material as demonstrated by flow cytometry with propidium iodide, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers had progressively decreased cellularity. The erythroblast populations as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoAΔ/Δ definitive erythroblasts also exhibited increased frequency of polyploidy. These polyploid cells were Ki67-negative by immunohistochemistry, indicating that they were not actively mitotic. Apoptosis was not significantly increased in the RhoA-deficient erythroblasts as determined by Annexin-V positivity. However, the late RhoAΔ/Δ erythroblasts demonstrated a significant increase in necrosis as determined by 7AAD and Annexin-V double-positivity (n=3, p<0.05). Increased phosphorylation of p53 was evident by immunoblotting, likely functioning as a polyploidy checkpoint after failure of abscission due to RhoA deficiency. Further evidence that RhoA is significant for microtubule organization in erythroblasts was attained by immuno-localization of RhoA at the microtubule ends and by decreased nuclear polarization in EpoR-Cre RhoAΔ/Δ orthochromatic erythroblasts, as shown by decreased delta centroid Ter119-Draq5 (the distance between the centers of the erythroblast and the nucleus) in multispectral high-speed cell imaging in flow. Phosphorylated myosin regulatory light chain (pMRLC) was found significantly decreased by immunoblotting of RhoAΔ/Δ fetal liver erythroblasts, indicating that RhoA controls phosphorylation of MRLC in erythropoiesis. Increased PAK phosphorylation was noted pointing towards compensatory upregulation of Rac GTPases in the RhoA-deficient cells, which was confirmed by quantitative RT-PCR. We further examined the erythroblastic islands in RhoAΔ/Δfetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, which has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010).Thus, RhoA GTPase regulates erythroblast differentiation and enucleation by dynamic coordination of the microtubule-actomyosin machineries to achieve successful abscission. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Our results reveal novel molecular components and cellular processes in erythropoiesis that may be important for improving the efficiency of red blood cell production in vitro. Disclosures:No relevant conflicts of interest to declare.

Contribution of the Local RAS to Hematopoietic Function: A Novel Therapeutic Target

The renin-angiotensin system (RAS) has long been a known endocrine system that is involved in regulation of blood pressure and fluid balance. Over the last two decades, evidence has accrued that shows that there are local RAS that can affect cellular activity, tissue injury, and tissue regeneration. There are locally active ligand peptides, mediators, receptors, and signaling pathways of the RAS in the bone marrow (BM). This system is fundamentally involved and controls the essential steps of primitive and definitive blood-cell production. Hematopoiesis, erythropoiesis, myelopoiesis, thrombopoiesis, formation of monocytic and lymphocytic lineages, as well as stromal elements are regulated by the local BM RAS. The expression of a local BM RAS has been shown in very early, primitive embryonic hematopoiesis. Angiotensin-converting enzyme (ACE-1, CD143) is expressed on the surface of hemangioblasts and isolation of the CD143 positive cells allows for recovery of all hemangioblast activity, the first endothelial and hematopoietic cells, forming the marrow cavity in the embryo. CD143 expression also marks long-term blood-forming CD34+ BM cells. Expression of receptors of the RAS is modified in the BM with cellular maturation and by injury. Ligation of the receptors of the RAS has been shown to modify the status of the BM resulting in accelerated hematopoiesis after injury. The aim of the present review is to outline the known functions of the local BM RAS within the context of primitive and definitive hematopoiesis as well as modification of BM recovery by administration of exogenous ligands of the RAS. Targeting the actions of local RAS molecules could represent a valuable therapeutic option for the management of BM recovery after injury as well as neoplastic disorders.

Erythroid Specific RhoA Deficiency Causes Dysplastic and Inefficient Fetal Erythropoiesis with Lethal Embryonic Anemia Due to Defects in Erythroblast Cytokinesis and Enucleation

Abstract 82RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis (Schwartz, J Cell Sci 2004). We recently demonstrated evidence that enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis. It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte. We showed that RhoA-related Rac GTPases organize actin in the actomyosin ring and aggregate lipid rafts in the furrow between nascent reticulocyte and pyrenocyte during enucleation (Konstantinidis et al, Blood 2012).Based on the resemblances between erythroblast enucleation and cytokinesis we hypothesized that RhoA dynamically controls erythroblast enucleation by molecular pathways analogous to the ones involved in cytokinesis. To define the mechanistic contribution of RhoA signaling in erythropoiesis and enucleation, we generated mice with erythroid specific deletion (EpoRGFPcre/+ driven) of RhoA. Erythroid-specific deletion of RhoA caused severe anemia, which was fatal in utero by E14.5. These embryos as well as their yolk sacs had a pallid, anemic appearance and their peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers at E12.5 had approximately 33% of the cellularity of WT littermates (n=9, p<0.001). Analysis of definitive erythropoiesis in the RhoAΔ/Δ fetal liver showed that despite decreased cellularity, the erythroid progenitors BFU-E and CFU-E were not different in total number per fetal liver. In contrast, the erythroblast populations, as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoA-deficient fetal liver erythroblasts expressed significantly higher levels of the α4 and α5 integrin subunits, suggesting a compensatory increase of the adhesion receptors due to impaired downstream signaling. We further examined the erythroblastic islands in RhoAΔ/Δ fetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were more loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, that has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010). Upon evaluation of the RhoA protein level and phosphorylated myosin regulatory light chain (pMRLC) by immunoblotting of RhoAΔ/Δ fetal liver cells, pMRLC was found significantly decreased, indicating that one of the signaling pathways regulated by RhoA in erythropoiesis involves phosphorylation of MRLC. Moreover, treatment of wild type erythroblasts in in vitro erythropoiesis assays with the pharmacological inhibitor Y27632 of Rho-associated protein kinase (ROCK), a downstream target of RhoA participating in myosin regulation, resulted in a decrease of enucleation by up to 38% (n=3, p<0.05).Thus, RhoA GTPase regulates erythroblast differentiation and enucleation via regulation of the actomyosin cytoskeleton by ROCK-mediated phosphorylation of MRLC. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Further exploration of the role of RhoA in erythropoiesis has the potential to reveal targets for therapeutic interventions for anemias due to terminal erythroid maturation defects as well as for improving the efficiency of red blood cell production in vitro. Disclosures:No relevant conflicts of interest to declare.

Identification of the Hemogenic Endothelial Progenitor and Its Direct Precursor in Human Pluripotent Stem Cell Differentiation Cultures

Hemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential for enabling the investigation of the molecular determinants of HSC specification. Here, we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by a VE-cadherin(+)CD73(-)CD235a/CD43(-) phenotype. This phenotype discriminates true HEPs from VE-cadherin(+)CD73(+) non-HEPs and VE-cadherin(+)CD235a(+)CD41a(-) early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post-primitive-streak stage of differentiation directly from VE-cadherin-negative KDR(bright)APLNR(+)PDGFRα(low/-) hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR(+)PDGFRα(+) mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal of facilitating the generation of HSCs from hPSCs.