Fetal Liver Hematopoietic Progenitor Research Articles

Histone methyltransferase SETDB1 safeguards mouse fetal hematopoiesis by suppressing activation of cryptic enhancers

The H3K9me3-specific histone methyltransferase SETDB1 is critical for proper regulation of developmental processes, but the underlying mechanisms are only partially understood. Here, we show that deletion of Setdb1 in mouse fetal liver hematopoietic stem and progenitor cells (HSPCs) results in compromised stem cell function, enhanced myeloerythroid differentiation, and impaired lymphoid development. Notably, Setdb1-deficient HSPCs exhibit reduced quiescence and increased proliferation, accompanied by the acquisition of a lineage-biased transcriptional program. In Setdb1-deficient HSPCs, we identify genomic regions that are characterized by loss of H3K9me3 and increased chromatin accessibility, suggesting enhanced transcription factor (TF) activity. Interestingly, hematopoietic TFs like PU.1 bind these cryptic enhancers in wild-type HSPCs, despite the H3K9me3 status. Thus, our data indicate that SETDB1 restricts activation of nonphysiological TF binding sites which helps to ensure proper maintenance and differentiation of fetal liver HSPCs.

Transduction of Human Fetal Liver Hematopoietic CD34+ Stem and Progenitor Cells into a Cell Line by Enhancing Telomerase Activity

Background: Human fetal liver hematopoietic stem cells have proven potential as therapeutics but lack extensive research due to their limited supply. Even in vitro expanded fetal liver hematopoietic stem cells enter senescence or lose their self-renewal capacity after a few days in culture. The present study aimed to obtain a homogeneous and persistent supply of hematopoietic stem cells from the fetal liver by establishing a cell line through immortalization of cells by enhancing telomerase activity. Materials and Methods: Human fetal liver hematopoietic CD34+ stem and progenitor cells were transformed and immortalized using retroviruses carrying the human telomerase (hTERT) gene. Following transduction, telomerase activity was assessed using the TRAP assay and telomere length was examined by Southern blotting in transduced cells. Their characterization was conducted using flowcytometry to analyze the CD34+ population of hematopoietic stem cells and their colony forming potential using colony forming unit (CFU) assay. Results: After transduction with hTERT, the life span of human fetal liver hematopoietic CD34+ stem and progenitor cells were extended to 80 population doublings, without any change in cell morphology or population doubling times. Constitutive hTERT expression enhanced the replicative capacity and prevented terminal differentiation of CD34+ fetal liver hematopoietic stem and progenitor cells (FLHSPCs). Moreover, hTERT-transduced stem cells maintained their telomere length and telomerase activity. Conclusion: By introducing telomerase activity into hematopoietic stem and progenitor cells, their lifespan can be extended while maintaining stemness. These modified cells hold promise for in vitro research focused on studying hematopoietic stem cells derived from fetal liver.

O-Linked N-Acetylglucosamine Transferase Ensures Survival of Mouse Fetal Liver Hematopoietic Progenitors Partly by Regulating Bcl-xL and Oxidative Phosphorylation.

O-linked N-acetylglucosamine transferase (OGT) critically regulates wide variety of biological processes such as gene expression, metabolism, stress response, signaling and proteostasis. In adult hematopoiesis, OGT is crucial for differentiation of B and T cells and the maintenance of hematopoietic stem cells (HSCs). However, a role for OGT in fetal liver (FL) hematopoiesis remains unknown. To investigate a role for OGT in FL hematopoiesis, we conditionally disrupted OGT in hematopoietic cells in developing FLs. Hematopoietic specific disruption of OGT resulted in embryonic lethality in late stage of gestation due to severe anemia and growth retardation. OGT loss led to profound reduction of differentiating erythroid cells and erythroid progenitors in FLs due to massive apoptosis. In addition, clonogenic capacity of FL cells was severely impaired by OGT loss. Interestingly, expression of BCL-XL, a well-known inhibitor of apoptosis in FL cells, dramatically decreased, and the levels of reactive oxygen species (ROS) were increased in OGT-deficient FL cells. Overexpression of Bcl-xL and reduction of ROS significantly restored the colony formation of OGT-deficient FL cells. This study revealed a novel role for OGT during embryogenesis, which ensures survival of FL hematopoietic cells partly by regulating Bcl-xL and oxidative phosphorylation.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

In vitro expansion of fetal liver hematopoietic stem cells

Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

Characterization of a Novel JAK1 Pseudokinase Mutation in the First Case of Trisomy 21-Independent GATA1-Mutated Transient Abnormal Myelopoiesis

Clonal proliferation of megakaryoblasts, called transient abnormal myelopoiesis (TAM), is a rare disease of newborns triggered by trisomy 21 (constitutional or somatic) together with acquired mutations of GATA1 resulting in the exclusive production of its short variant - GATA1s. No other TAM drivers have been described so far. We have diagnosed a unique TAM case with a typical clinical and laboratory manifestation but without the gain (or any other aberration) of chromosome 21. Thorough genomic profiling revealed 4 somatic mutations: GATA1 D65_C228del, JAK1 F636del, FN1 R2420C and SPIRE2 R471W. With respect to the generally accepted 2-hit theory, we hypothesized that this TAM arose from a collaboration of the atypical GATA1 mutation (not inducing GATA1s) with (at least) one of the other identified mutations. Unlike SPIRE2 and FN1 aberrations, various mutations of the JAK1 kinase have been previously described as leukemia drivers, suggesting JAK1 F636del as a top candidate for the second hit. Moreover, JAK1 mutations have been associated with the transformation of TAM into acute megakaryoblastic leukemia (Nikolaev et al., Blood, 2013). The aim of our project was to functionally characterize this novel JAK1 mutation. Phenylalanine 636 belongs to a phylogenetically conserved triad of amino acids suggested to control catalytic activity of JAK1 via mediating a switch between the supposedly active and inactive conformations (Toms et al., Nat Struct Mol Biol, 2013). Hence, F636 seems to be essential for JAK1 function. Surprisingly, homology modeling showed that loss of F636 is compatible with both functionally opposite conformations. Indeed, Western blot analysis of JAK/STAT signaling in transiently transformed HEK293T cells showed that catalytic activity is preserved in JAK1 F636del. However, we observed lower levels of auto- and STATs- phosphorylation compared to wild-type (wt) JAK1 suggesting decreased kinase activity of JAK1 F636del. Subsequently, we tested the oncogenic potential of JAK1 F636del in the Ba/F3 cell assay; unlike the known oncogenic JAK1 variant (JAK1 V658I), JAK1 F636del did not induce IL3-independent growth. To further assess phenotypic impact of F636del, we introduced JAK1 F636del into murine bone marrow and fetal liver hematopoietic stem and progenitor cells (HSPCs) using lentiviruses and performed colony forming assays. The number and morphology of colonies did not differ in JAK1 F636del compared to wt JAK1. Furthermore, we assessed the impact of JAK1 F636del in the context of mutated GATA1. We utilized the in-vitro model recently described by Labuhn et al. (Cancer Cell, 2019), in which the CRISPR/Cas9-mediated induction of Gata1s expression leads to the expansion and sustained proliferation of fetal liver HSPCs from embryonic day 13.5 ROSA26:Cas9-EGFPki/wt mice. Similar to wt JAK1, lentivirally introduced JAK1 F636del had no impact on the proliferation and maturation status of such Gata1-edited HPSCs irrespective of the timing of its introduction (simultaneously with Gata1 editing versus into fully established Gata1-edited culture) or of culturing conditions (fully cytokine-supplemented growth-supportive versus cytokine-depleted growth-restrictive medium). Altogether, we show that unlike known oncogenic variants, F636del identified in the first case of trisomy 21-independent TAM attenuates JAK1 kinase activity. The results of our phenotypic studies question the potential contribution of this mutation to TAM development. Interestingly, Labuhn et al. (2019) recently showed that non-activating JAK mutations occur at higher than random frequency in trisomy 21-dependent TAM. This tempts us to speculate that JAK1 mutations may still play a role in TAM. Yet, this role may significantly differ from that of known oncogenic mutations; it may result from attenuation/modulation instead of activation of downstream signaling and it may remain unrevealed utilizing the currently available sophisticated, yet still imperfect experimental models. Support: GAUK 86218, EHA Research Mobility Grant Disclosures No relevant conflicts of interest to declare.

SIX1 Is a Novel Effector Gene in CALM-AF10 Leukemia

Background: The CALM-AF10 translocation is detected in ~10% of T-cell acute lymphoblastic leukemias (T-ALLs), and in some acute myeloid leukemias (AMLs). CALM-AF10 leukemias are characterized by high expression of proleukemic HOXA genes, which serve a critical role in hematopoiesis. We hypothesized that identification of novel CALM-AF10 effector genes may yield new therapeutic targets in this difficult to treat leukemia. We took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal (NES) in CALM. Using next generation sequencing, we determined that, SIX1, similar to HOXA genes, is increased in CALM-AF10 leukemias and decreased in response to CRM1 inhibition with Leptomycin B (LMB). Design/Methods: RT-qPCR and Chromatin Immunoprecipitation were performed using both bone marrow progenitors and murine embryonic fibroblasts (MEFs) transduced with CALM-AF10 or an empty vector, with and without LMB. The ability of SIX1 to enhance self-renewal of hematopoietic progenitors was examined by measuring the colony-forming ability of transduced fetal liver hematopoietic progenitor cells. CRISPR-Cas9 was used to silence SIX1 in Human Embryonic Kidney 293 (HEK293) cells. Results: RT-qPCR confirmed overexpression of SIX1 in both CALM-AF10 transduced MEFs and CALM-AF10 leukemias, with decreased SIX1 expression observed in the presence of LMB. ChIP analysis showed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver cells was sufficient to increase the self-renewal potential of colony-forming progenitors. SIX1 was successfully knocked out in HEK293 cells without a significant effect on HEK293 proliferation. Conclusions: The SIX1 homeobox gene is highly expressed during development and its expression is silenced post-embryogenesis. Increased SIX1 expression has been reported in numerous solid tumors. We have determined that SIX1 is upregulated in CALM-AF10 leukemias, and increases the self-renewal potential of hematopoietic progenitors. Using CRISPR-Cas9 to silence SIX1, we have demonstrated that SIX1 is not essential for cell survival, and that its inhibition may impair CALM-AF10 leukemia cell proliferation. Thus, SIX1 may play a pathogenic role in leukemogenesis and is a potential therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

Unraveling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis.

B-cell acute lymphoblastic leukemia is the commonest childhood cancer. In infants, B-cell acute lymphoblastic leukemia remains fatal, especially in patients with t(4;11), present in ~80% of cases. The pathogenesis of t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemia remains difficult to model, and the pathogenic contribution in cancer of the reciprocal fusions resulting from derivative translocated-chromosomes remains obscure. Here, “multi-layered” genome-wide analyses and validation were performed on a total of 124 de novo cases of infant B-cell acute lymphoblastic leukemia uniformly diagnosed and treated according to the Interfant 99/06 protocol. These patients showed the most silent mutational landscape reported so far for any sequenced pediatric cancer. Recurrent mutations were exclusively found in K-RAS and N-RAS, were subclonal and were frequently lost at relapse, despite a larger number of non-recurrent/non-silent mutations. Unlike non-MLL-rearranged B-cell acute lymphoblastic leukemias, B-cell receptor repertoire analysis revealed minor, non-expanded B-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cell acute lymphoblastic leukemias and the most immature human fetal liver hematopoietic stem and progenitor cells, confirming a “pre-VDJ” fetal cellular origin for both t(4;11) and RASmut. The reciprocal fusion AF4-MLL was expressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA cluster genes are exclusively expressed in AF4-MLL-expressing patients. Importantly, AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs. 11.7%, P=0.001), and overall survival (73.7 vs. 25.2%, P=0.016). AF4-MLL expression retained its prognostic significance when analyzed in a Cox model adjusting for risk stratification according to the Interfant-06 protocol based on age at diagnosis, white blood cell count and response to prednisone. This study has clinical implications for disease outcome and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lymphoblastic leukemia.

U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS.In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal.Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. DisclosuresNo relevant conflicts of interest to declare.

A novel prospective isolation of murine fetal liver progenitors to study in utero hematopoietic defects.

In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers’ expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible.

Mice expressing KrasG12D in hematopoietic multipotent progenitor cells develop neonatal myeloid leukemia.

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that bears distinct characteristics associated with abnormal fetal development. JMML has been extensively modeled in mice expressing the oncogenic KrasG12D mutation. However, these models have struggled to recapitulate the defining features of JMML due to in utero lethality, nonhematopoietic expression, and the pervasive emergence of T cell acute lymphoblastic leukemia. Here, we have developed a model of JMML using mice that express KrasG12D in multipotent progenitor cells (Flt3Cre+ KrasG12D mice). These mice express KrasG12D in utero, are born at normal Mendelian ratios, develop hepatosplenomegaly, anemia, and thrombocytopenia, and succumb to a rapidly progressing and fully penetrant neonatal myeloid disease. Mutant mice have altered hematopoietic stem and progenitor cell populations in the BM and spleen that are hypersensitive to granulocyte macrophage-CSF due to hyperactive RAS/ERK signaling. Biased differentiation in these progenitors results in an expansion of neutrophils and DCs and a concomitant decrease in T lymphocytes. Flt3Cre+ KrasG12D fetal liver hematopoietic progenitors give rise to a myeloid disease upon transplantation. In summary, we describe a KrasG12D mouse model that reproducibly develops JMML-like disease. This model will prove useful for preclinical drug studies and for elucidating the developmental origins of pediatric neoplasms.

Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells.

Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at the single cell level, through subsequent transplantation and high-throughput sequencing. Furthermore, cellular barcoding allows for bona fide hematopoietic stem cells (HSCs) to be defined based on functional rather than immunophenotypic parameters. This protocol describes the work flow of lentiviral cellular barcoding, tracking 14.5 days post coitum (d.p.c.) fetal liver (FL) Lineage-Sca+cKit+ (LSK) HSPCs following long-term reconstitution (Figure 1) ( Kristiansen et al., 2016 ), but can be adapted to the cell type or time frame of choice. Figure 1.Summary of experimental workflow ( Naik et al., 2013 ).

Replication Associated Stress in the Fetal Stem Cell Pool of FA Mice Can be Rescued By Pharmacologic Inhibition of Constitutively Activated P38 MAPK

Fanconi Anemia (FA) is a rare, recessively heritable disorder with prominent failure of hematopoiesis. The physiologic role of FA proteins has not been fully resolved to date. While several existing model systems delineate its role in DNA damage response caused by alkylating agents, aldehydes, and inflammatory cytokines, all rely on experimental induction. We previously demonstrated the in utero onset of hematopoietic failure in mice with genetic disruption of Fancc. Herein, we found significant deficits in the fetal liver (FL) hematopoietic stem and progenitor cell (HSPC) pool in Fancd2 mice. Both AA4.1+ Sca-1+ Lin- expressing progenitors (ASL) and CD48- CD150+ Lin- Sca-1+ (SLAM) cells were decreased in frequency in Fancd2-/- versus WT FL. Similarly, we observed a significant decrease in progenitor colony formation and deficits in primary and secondary transplantation among Fancd2-/- FL compared to WT. Fancd2-/- FL cells were characteristically sensitive to mitomycin C and had significantly fewer SLAM cells in the G0 phase of cell cycle and elevated p21 expression, indicating canonical P53 activation. Consistent with prior reports by other groups on embryonic stem cells and our own Fancc-/- FL studies, we found neither exaggerated frequency of apoptotic cells, nor transcriptional induction of Puma or Noxa. We hypothesized that the observed deficits in developmental HSPC pool expansion reflect replication-associated stress. At the transcriptional level, we found activation of the DNA damage response via Rad51 and Prkdc, corroborated by immunofluorescent imaging of Rad51 foci as well as comet assays in FL cells. Next, we tested P38 MAPK as a stress response previously found to confer repopulation deficits in postnatal BM failure among Fancc and Fanca mice; here, our experiments revealed baseline (unprovoked) activation of phospho-p38 and rescue of Fancd2-/- progenitor colony formation using a pharmacological inhibitor, SB203580. Results were further strengthened by transplantation, revealing increased Fancd2-/- donor chimerism after in vivo administration of SB203580. The gains in donor chimerism persisted even after cessation of drug administration. These results suggest that replication-associated stress in the rapidly cycling fetal Fancd2-/- HSPC pool evokes a cellular stress response that constrains physiological expansion. Our work emphasizes the prenatal onset of hematopoietic failure and reveals pharmacological rescue by inhibition of constitutively active P38 MAPK. Furthermore, FA fetal hematopoiesis is an original model of unprovoked hematopoietic failure that allows the study of physiologic role of FA proteins in HSPC. DisclosuresNo relevant conflicts of interest to declare.

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis.

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.

MicroRNA-486-5p is an erythroid oncomiR of the myeloid leukemias of Down syndrome

AbstractChildren with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS.

EBF Deficient Hematopoietic Progenitor Cells Potentialy Differentiated into Immature B Cell: EBF1 Dispensable for B Cell Linage Commitment from Pro-B to Pre-B Stage

Introduction: Canonical notion demonstrates that Cell fate is determined by transcriptional factor. Accordingly, the lineage specific transcriptional factors have been investigated for various kinds of cells. Especially, in the hematopoietic system, the extensive research for lineage specific transcriptional factors had elucidated the transcriptional factors which regulated lineage commitment. B cell commitment and development requires the activities of multiple transcription factors, including the early B cell factor (EBF), PAX5, and E2A. These transcription factors regulate B cell development in a stage specific manner. The hematopoietic progenitor cells which are deficient for any of them, cannot commit B cells. Among them EBF1 is presumed to be more potent. Rescue early pro B cell to induce the expression of several key proteins including RAG that enable gene rearrangement to occur by opening of IgH locus.We found that the B cell developmental arrest caused by EBF1 deficiency can be rescued by a single non coding RNA. These B cells which are deficient EBF1 but showed the expression of CD19, B cell lineage specific surface marker and VDJ recombination, molecular markers of B cell commitment in vitro B cell differentiation system cocultured with Tst4 cells, stromal cell lines. We further investigated the quality and differentiation potential of these B lineage commitment cells in the in vivo mouse model and elucidated the mechanism of this phenomenon.Material and methods: We collected EBF1−/− fetal liver hematopoietic progenitor (Lin−) cells and cultured them on TSt-4 stromal cells after infecting with non coding RNA and control vector in IMDM medium containing cytokines and then injected it into NOG mice. Collect bone marrow (BM), thymus and spleen from those mice. Then comprehensive Gene-Expression analysis, real time PCR for VDJ recombination analysis was performed and checked surface marker by flowcytometry.Result: We analyzed BM and spleen of non coding RNA infected EBF1 KO cells injected mice and found the expression of CD19 in BM as well as in spleen and upregulated of B220 also, comparing with control vector expressed cells. Furthermore, surface IgM expression of CD19 positive cells in the spleen is upregulated compared with the cells in the BM (Figure 1). Several target genes of the non-coding RNAs were identified by use of cDNA array analysis and luciferase reportor assay. Among them, several genes were involved in TGF beta pathway. As TGF beta family and the pathway, has been reported a critical factor which is negatively regulating B lymphopoiesis (Figure 2). We hypothesized that TGF beta family genes such as Tgfβr3, Acvr2a, are responsible for B cell differentiation for which EBF1 is dispensable .We cultured EBF KO cells for 14 days with and without TGF beta 1,2,3 antibody and Activin A antibody on TST4 cells. We found that increase mean intensity (MFI) of B220 into antibodies positive cocultured cells (Figure 3) to suggest, the suppression of TGF beta pathway is partially responsible for B cell differentiation under EBF1 deficiency.Conclusion: Canonical notion of cell fate determination of B cells defines that EBF1 is an indispensible factor for B lymphopoiesis. However, from our previous and present study it is proved that without EBF1 B cell development can progress to pro B to pre B cell and Immature B cell stage and “VDJ recombination” occur in the absence of EBF. Furthermore, in vivo mouse model, EBF1 deficient hematopoietic progenitor cells differentiated into IgM positive cells. Therefore we can conclude that EBF is dispensable of VDJ recombination, opening of IgH locus, binding of RAG protein and B cell differentiation to the mature stage. One of the mechanisms is possibly due to the stimuli from microenvironment, such as TGF beta family and pathway. Furthermore, the detail mechanism of IgH locus opening, epigenetic changes and chromatin remodeling around the IgH locus in the absence of EBF is under investigation. [Display omitted] [Display omitted] [Display omitted] DisclosuresChanda:Japan Society for the Promotion of Science(JSPS): Research Funding.

Metabolic Programming of the Fetal Liver Hematopoietic Stem and Progenitor Cell Pool

Evidence in several organ systems demonstrates that pregnancy presents a window of vulnerability for establishing a foundation for health or chronic disease. Overnutrition and the complex metabolic changes that can accompany it can result in permanent phenotypic changes and a predisposition to metabolic syndrome, inflammatory or immune-mediated diseases. We previously reported that prenatal overnutrition stunted fetal liver size. Herein, we hypothesize that this might perturb hematopoietic stem and progenitor cell (HSPC) expansion.To test the effects of a high-fat diet (HFD) and maternal obesity on offspring hematopoiesis, we used a mouse model of diet-induced obesity, feeding female mice a HFD or control diet starting at 5-7 weeks of age and keeping them on the respective diet during subsequent breeding and pregnancy. We then studied offspring at gestational day 14.5 by immunophenotyping, gene expression analysis, qRT-PCR, and transplantation. Fetal livers from HFD offspring had 51% fewer c-Kit+ Sca-1+ Linlo/- and 27% fewer AA4.1+ Sca-1+ Linlo/- (ASL) hematopoietic stem and progenitor cells (HSPC) than controls. This restriction in HSPC numbers was not due to apoptosis or increased reactive oxygen species, as tested by flow cytometry.To determine whether there might be an increase in hematopoietic differentiation to account for relative HSPC deficiencies in HFD livers, we examined hematopoietic lineage subsets. HFD fetal livers had a relative increase in myeloid (Gr-1+/Ter119+) and B220+ lymphoid cells, with comparable proportion of CD3+ cells to controls. Taken together, these results suggest that chronic HFD fetal programming skews fetal liver HSPCs toward differentiation.When we examined global gene expression of male HFD fetal livers versus controls by RNA-seq, we found differential expression of 125 genes. Among the upregulated transcripts, several were involved in hematopoietic regulation, stress response, and HSPC migration. We then used qRT-PCR to test for expression of several of these genes, along with genes critically involved in fetal HSPC self-renewal, within an HSPC-enriched (Sca-1+) population of chronic HFD fetal liver cells. As in RNA-seq, Matrixmetalloproteinase-8 and 9 (Mmp8, Mmp9), which are involved in cell mobilization, were upregulated in HFD-programmed cells. Early growth response-1 (Egr-1) was downregulated as well, further suggesting premature migration of HSPCs from HFD fetal liver. Hmga2, which is implicated in fetal stem cell self-renewal, and its direct target, Igf2bp2, were significantly downregulated in chronic HFD Sca-1+ cells. Along with the immunophenotyping data, these findings suggest that maternal obesity and HFD bias HSPCs toward differentiation, at the expense of self-renewal. To dissect the direct metabolic impact, we studied fetal livers from timed pregnancy cohorts after acute HFD exposure or diet reversal in obese dams, which partially ameliorated several molecular and immunophenotypic endpoints.Finally, we performed a functional test of chronic HFD fetal liver cells by transplantation. A non-competitive transplant into irradiated male recipients yielded no difference in chimerism between HFD or control fetal liver-engrafted animals. Next, we preconditioned a cohort of female and male animals on HFD (or control diet) for 11 weeks, irradiated them, and then competitively transplanted them with a 1:1 ratio of HFD and control fetal liver cells. HFD-programmed fetal liver HSPCs engrafted HFD-conditioned male recipients at significantly lower rates than in HFD-conditioned female or control recipients of either sex. HFD-programmed donor cells retained the significant bias toward the myeloid (Gr-1+/Mac-1+) lineage, noted in the primary graft cells, and away from the B220+ B cell lineage in HFD-conditioned males.In aggregate, prenatal HFD and maternal obesity suppress self-renewal in favor of HSPC differentiation during a time of critical developmental expansion. This suggests an HSPC defect that appears at least partly specified by the stem cell microenvironment. Our work is the first to demonstrate metabolic vulnerability of the hematopoietic stem and progenitor cell compartment and establishes the hematopoietic system as a target for in utero developmental programming. DisclosuresNo relevant conflicts of interest to declare.

Nestin+ Pericytes In The Fetal Liver Are Necessary To Maintain HSCs

Although most hematopoietic stem cells (HSCs) are quiescent under homeostasis in the adult bone marrow, they are actively proliferating during development. Definitive HSCs, marked by the ability to repopulate a lethally irradiated adult mouse, are first detectable in the aorta-gonad-mesonephros region around E10.5, and then colonize the fetal liver (FL) to expand in this organ until E15 when hematopoietic activity shifts to the fetal bone marrow. The role of the microenvironment, or niche, in the regulation of HSCs in the FL, a site of physiological expansion, is unclear. Fetal liver sinusoidal cells as well as hepatic progenitors have been proposed as sources of supportive signals that drive the exponential expansion of HSCs, however, exact cell type(s) supporting HSC expansion have not been defined. Since stromal cells marked by Nestin-GFP form niches for HSCs in the BM, we have hypothesized that similar cells exist in the FL. Indeed, a rare population of Nestin+ cells (CD31-CD45-Ter119-) comprising 0.02 ± 0.01% of nucleated FL cells was isolated. FL Nestin+ cells express surface markers similar to their BM counterparts, including PDGFRα, CD51, and endoglin. Since BM Nestin+ cells are enriched in mesenchymal stem and progenitor cell (MSPC) activity, we next tested this activity in FL Nestin+ cells. We found that the entire CFU-F forming capacity of the FL was contained within FL Nestin+ cells, further suggesting similarities of these cells between these two hematopoietic organs. In addition, FL Nestin+ cells were enriched for the HSC maintenance genes SCF and CXCL12, suggesting that they may also serve as the HSC niche in the FL. FL Nestin+ cells were localized on the abluminal side of large-bore arteries and expressed the pericyte markers, α-smooth muscle actin and NG2. To investigate the function of the FL Nestin+NG2+ cells in vivo, we generated a genetic depletion mouse model of NG2+ cells (NG2-cre / inducible diphtheria toxin-A (iDTA) transgenic mice). We found that E14.5 NG2-Cre;iDTA mouse embryos developed normally compared to fl-DTA control littermates. The frequency and absolute numbers of HSCs per FL, however, were reduced by about 40% in NG2-Cre;iDTA embryos (control / depleted; 0.01146 ± 0.0008% / 0.00660 ± 0.0008% of nucleated cells, p=0.0015; 606 ± 76 / 377 ± 48 HSCs/FL, p=0.03, , N=6-7 per group) suggesting that Nestin+ cells are required to maintain HSCs/progenitors in vivo. To interrogate further the relationship between FL Nestin+ cells and HSCs, we adopted the re-aggregate organ culture assay (Sheridan, Genesis 2009). Sorted lineage- FL hematopoietic progenitor cells and FL parenchymal cells either with or without sorted Nestin+ cells were re-aggregated and cultured for 7 days in serum-free, cytokine-free, media. After 7 days of culture, we found that CD150+ CD48− CD41− Lineage− HSCs were maintained in re-aggregates containing Nestin+ cells, but not when Nestin+ cells were absent. To confirm that FL Nestin+ cells were essential to maintain functional HSCs, re-aggregated cells cultured for 7 days were transplanted together with competitor bone marrow into lethally irradiated mice. We found that the contribution to the peripheral blood at 8 weeks post-transplant was only observed in the re-aggregated group containing Nestin+ cells. These preliminary data indicate that factors derived from FL Nestin+ cells are required to maintain HSCs in re-aggregate cultures. These findings suggest that HSCs inhabit similar microenvironments in temporally and spatially distinct hematopoietic organs. Further studies on differences between Nestin+ cells in these tissues may shed light on the mechanisms that determine the finely tuned quiescence, self-renewal and differentiation of HSCs. Disclosures:No relevant conflicts of interest to declare.

The RUNX1-Regulated Cebpa +37 Kb Enhancer Directs Human CD4 Transgene Expression To Long-Term Hematopoietic Stem Cells and Preferentially To Myeloid Compared With Lymphoid Or Erythroid Progenitors

We found that adult mice lacking RUNX1 have increased monopoiesis relative to granulopoiesis correlated with reduce Cebpa RNA expression in myeloid progenitors and that RUNX1 binds and activates Cebpa reporters via two sites in the Cebpa promoter and four conserved sites in a 450 bp +37 kb enhancer (Guo et al, Blood, 2012). This enhancer interacts with p300 and contains the H3K4me1 enhancer mark in myeloid cells. Previously reported ChIP-Seq data had demonstrated interaction of RUNX1 with the +37 kb enhancer, along with GATA2, SCL, Gfi-1b, MEIS1, Fli-1, ERG, PU.1, Lyl1, and LMO2. We have now generated transgenic C57BL/6 mice in which the Cebpa +37 kb enhancer positioned upstream of its 845 bp promoter (-720 to +125) directs expression of cytoplasmically truncated human CD4 (hCD4). Two founders with germ line transmission have been identified – both have detectable hCD4 RNA in marrow, at approximately 10-fold increased levels in one line compared to the other, with hCD4 expression detectable by FACS only in the higher expressing line. Analysis of 4-7 mice reveals that hCD4 expression is detected only in a small subset of mature hematopoietic cells, present in 6% of Mac-1+Gr-1+ neutrophils, 31% of Mac-1+Gr-1- monocytes, 4% of CD3+ T cells, 3% of CD19+ or 7% of B220+ B-cells, and 5% of Ter119+ erythroid cells. 6.5+/-1.9% of total marrow mononuclear cells and 13+/-4% of Lin- marrow cells express hCD4. At the progenitor level, hCD4 was evident in 56+/-7% of CMP, 70+/-5% of GMP, 36+/-9% of CLP, and only 1.5+/-1.0% of MEP. There was>100-fold enrichment of myeloid CFU but a 2-fold decrease in B-lymphoid colonies comparing hCD4+ versus hCD4- fractions of total marrow. Staining LSK cells for CD34 and FcγR demonstrated that 91+/-10% of multi-potent progenitors (MPP), 61+/-8% of ST-HSC, and 17+/-8% of LT-HSC express hCD4. Similarly, staining for the SLAM markers demonstrated that 19+/-9% of LSK-CD48-CD150+ LT-HSC express hCD4. Mean fluorescence index of hCD4 staining was about 2-fold higher in LSK, MPP, ST-HSC, CMP, and GMP compared with CLP and LT-HSC and about 3-fold higher than mature blood myeloid cells. 2E6 hCD4+ or hCD4- CD45.2 marrow cells were transplanted into lethally irradiated CD45.1 syngeneic recipients. 5/5 mice receiving hCD4- cells died at 2 weeks, reflecting lack of even progenitor engraftment, whereas 5/5 mice receiving hCD4+ cells were alive at 20 weeks, reflecting expression of the transgene in functional LT-HSC, with secondary transplanted mice viable at 4 week to date. The marrow cells of surviving mice were almost exclusively CD45.2, with hCD4+ expression in Lin- or Lin-c-kit+ marrow cells similar to that of donor mice. Although our second founder does not allow hCD4 detection by FACS, analysis of hCD4 RNA in marrow stem and progenitor subsets for this line is in progress, as is competitive transplantation of hCD4- versus hCD4+ marrow cells from the higher-expressing line. Murine E13.5 fetal liver hematopoietic stem and progenitor cells express hCD4 in a pattern similar to adult marrow. In zebrafish, the murine Cebpa +37 kb enhancer directs ZsGreen expression specifically to the caudal hematopoietic territory (CHT), analogous to the murine fetal liver, at 72 hour post-fertilization. Murine Cebpa +37 kb enhancer:ZsGreen transgene expression, lacking the Cebpa promoter, overlaps in the CHT with that of murine Runx1 +23 kb enhancer:mCherry in compound transgenic fish. The Cebpa and Runx1 enhancers direct similar tdTomato expression from a Sleeping Beauty vector in the K562 cell line, and these vectors are being compared for expression in hESC derived hematopoietic cells. In summary, the Cebpa gene contains an enhancer active in both LT-HSC and myeloid progenitors. Further characterization of its regulation may provide insights into the development of these hematopoietic stem and progenitor subsets and into how Cebpa expression is impaired in myelodysplastic syndromes or in acute myeloid leukemia. In addition, the Cebpa enhancer may prove useful in efforts to optimize LT-HSC formation from hESC. Disclosures:Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Genome-wide mapping of histone modifications mediated by the polycomb-group complexes in fetal liver and adult bone marrow hematopoietic progenitor cells

Genome-wide mapping of histone modifications mediated by the polycomb-group complexes in fetal liver and adult bone marrow hematopoietic progenitor cells