Fetal HSCs Research Articles

Generation of complex bone marrow organoids from human induced pluripotent stem cells

The human bone marrow (BM) niche sustains hematopoiesis throughout life. We present a method for generating complex BM-like organoids (BMOs) from human induced pluripotent stem cells (iPSCs). BMOs consist of key cell types that self-organize into spatially defined three-dimensional structures mimicking cellular, structural and molecular characteristics of the hematopoietic microenvironment. Functional properties of BMOs include the presence of an in vivo-like vascular network, the presence of multipotent mesenchymal stem/progenitor cells, the support of neutrophil differentiation and responsiveness to inflammatory stimuli. Single-cell RNA sequencing revealed a heterocellular composition including the presence of a hematopoietic stem/progenitor (HSPC) cluster expressing genes of fetal HSCs. BMO-derived HSPCs also exhibited lymphoid potential and a subset demonstrated transient engraftment potential upon xenotransplantation in mice. We show that the BMOs could enable the modeling of hematopoietic developmental aspects and inborn errors of hematopoiesis, as shown for human VPS45 deficiency. Thus, iPSC-derived BMOs serve as a physiologically relevant in vitro model of the human BM microenvironment to study hematopoietic development and BM diseases.

Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids to Model Hematopoietic Development

The human bone marrow niche plays an essential role in supporting and regulating hematopoiesis throughout life and inherited bone marrow failure syndromes are often linked to dysfunction of the niche. Recently, methods for generating induced pluripotent stem cell (iPSC)-derived hematopoietic organoids (Motazedian et al., 2020) and bone marrow organoids (Khan et al., 2022) have been developed. We recently presented our approach to generate a model of a complex human bone marrow microenvironment in vitro (ASH annual meeting 2022 ). Here, we characterize our model in greater detail and analyse functionality of bone marrow organoid (BMO)-derived HSPCs. We devised a feeder- and serum-free protocol to generate a complex human iPSC-derived BMO within three weeks. In comparison to the protocol by Khan et al., we incorporated a sequential combination of Wnt activation and Activin/Nodal inhibition to induce mesoderm formation and patterning. We replicated the method with five different iPSC lines, including fibroblast- and PBMC-derived iPSCs, as well as iPSCs generated from renal epithelial cells isolated from urine samples. Using confocal and two-photon microscopy we visualized the spatial architecture of differentiated BMOs and detected CD45 + cells embedded into a network of CD31 + vascular cells and CD271 + stromal cells. PDGFRβ expressing pericytes were enwrapping the vascular cells. Moreover, stromal cells expressed CXCL12, Leptin receptor and Nestin. Electron microscopy, HE staining of organoid sections and 3D surface rendering showed lumen-forming vascular structures containing hematopoietic cells. To further elucidate the cell type composition, we performed single-cell RNA sequencing (scRNA-seq) of differentiated BMOs. Clustering of the scRNA-seq data by marker gene expression yielded three main populations: Cells in the endothelial cluster expressed canonical genes of arterial ECs. In the mesenchymal cluster we could distinguish pericytes, vascular smooth muscle cells and osteochondrogenic precursors. In the hematopoietic compartment, we identified cells expressing molecular signatures of granulocytes, monocytes and their progenitors, but also megakaryocytic-like cells and lymphoid progenitors. Remarkably, a cluster of cells expressed genes of fetal HSCs. This suggests that BMOs may recapitulate definitive hematopoiesis. To test T-cell differentiation potential in vitro, we sorted CD34 + HSPCs from dissociated BMOs and differentiated them within an artificial thymic organoid system (ATO). After three weeks we detected CD45 +CD4 +CD8 + cells, some of them expressing CD3 +TCRαβ +. Thus, we postulate that T-cells phenotypically resembling conventional T-cells emerge from BMO-derived HSPCs. To assess functional properties of BMO-derived HSPCs in vivo, we transplanted sorted CD34 + HSPCs into immunodeficient NSG mice and are currently analysing their engraftment potential. In summary, we provide a novel bone marrow niche model to study hematopoietic development in a complex three-dimensional organoid derived from human iPSCs.

Bclaf1 promotes hematopoietic stem cell repopulating capacity and self-renewal

Abstract Hematopoietic stem cells (HSC) balance differentiation and self-renewal to promote maintenance of mature blood cell lineages. The mechanisms that regulate HSC expansion during fetal hematopoietic development and adult HSC maintenance remain significant knowledge gaps. BCLAF1 (Bcl2-associated factor 1), a transcriptional regulator, is highly expressed in hematopoietic cells. We recently defined its function as a modulator of ETS-family transcription factor activity in developing B cells. Our objective in this study is to determine the molecular mechanism of BCLAF1 in regulation of fetal and adult HSCs. We used mouse models with selective deletion of Bclaf1 in hematopoietic cells to test whether BCLAF1 regulates HSC development and function. We find that loss of Bclaf1 results in significantly reduced numbers of fetal HSCs. The reduced HSC numbers persist in adult mice; but there is no additional decline or development of bone marrow failure with aging. In competitive transplant experiments, Bclaf1-deficient fetal and adult HSCs have defective repopulation and self-renewal capacity resulting in reduced reconstitution of all hematopoietic populations. Bclaf1-deficient HSCs do not have abnormalities in emergence, proliferation, cell death, or homing activity. Using single cell RNA-sequencing of fetal HSCs, we find that loss of BCLAF1 results in reduction of a subpopulation of HSCs and upregulation of the early response genes Jun, Fos, and Egr1. Collectively, these findings suggest BCLAF1 restrains activity of immediate early genes to preserve HSC self-renewal capacity and promote HSC expansion during both fetal development and repopulation of hematopoiesis after HSC transplant. Supported by grants from Children's Discovery Institute, St. Louis Children's Hospital Foundation, Siteman Cancer Center, and NIH (T32 AI007163).

Generation of reconstituted hemato-lymphoid murine embryos by placental transplantation into embryos lacking HSCs

In order to increase the contribution of donor HSC cells, irradiation and DNA alkylating agents have been commonly used as experimental methods to eliminate HSCs for adult mice. But a technique of HSC deletion for mouse embryo for increase contribution of donor cells has not been published. Here, we established for the first time a procedure for placental HSC transplantation into E11.5 Runx1-deficient mice mated with G1-HRD-Runx1 transgenic mice (Runx1-/-::Tg mice) that have no HSCs in the fetal liver. Following the transplantation of fetal liver cells from mice (allogeneic) or rats (xenogeneic), high donor cell chimerism was observed in Runx1-/-::Tg embryos. Furthermore, chimerism analysis and colony assay data showed that donor fetal liver hematopoietic cells contributed to both white blood cells and red blood cells. Moreover, secondary transplantation into adult recipient mice indicated that the HSCs in rescued Runx1-/-::Tg embryos had normal abilities. These results suggest that mice lacking fetal liver HSCs are a powerful tool for hematopoiesis reconstruction during the embryonic stage and can potentially be used in basic research on HSCs or xenograft models.

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

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

3151 – HETEROGENEITY OF EMERGING HSC PRECURSORS IN THE AGM REGION

Precursors of hematopoietic stem cells, termed pre-HSCs, are intermediate precursors during the maturation process from hemogenic endothelial cells (HECs) to HSCs in the aorta-gonad-mesonephros (AGM) region of the mouse embryo at E10.5-11.5. Pre-HSCs acquire an efficient adult-repopulating ability only after ex vivo co-culture, which precludes understanding of their native hematopoietic capacity. Our group recently identified the native repopulating ability of the pre-HSC population using immunodeficient neonatal mice that permit engraftment of embryonic hematopoietic precursors. We found that a freshly isolated pre-HSC population exhibited significantly greater B-1 lymphocyte-biased repopulating capacity than multi-lineage repopulating capacity. In addition, out lineage tracing mouse model labeling cdh5+ ECs by tamoxifen injection at various embryonic time points suggests that HECs at E9.5-10.5 produce HSCs and B-1 lineage cells simultaneously, while later HECs at E11.5 predominantly produce HSC and minimum B-1 lineage. The HSC-labeling using Fgd5CreERT2 mice also shows the minimum contribution of B-1a cell production of fetal liver HSCs at E14.5. To understand this unexpected B-1 lymphocyte bias of the pre-HSC population and acquisition of multi-lineage potential during commitment to the HSC fate, we performed single-cell RNA-sequencing (scRNA-seq) using E11.5 pre-HSCs, and E12.5 and E14.5 Fetal liver HSCs. The velocity analysis of the scRNA-seq data indicates the presence of a divergent point into two directions: features of HSPC and B-lymphoid progenitors. Our results display the cellular landscape of HSC and B-1 cell development from HECs and heterogeneity of the pre-HEC population.

INHIBITION OF LYSINE-SPECIFIC DEMETHYLASE 1A (LSD1) SUPPORTS HUMAN HSCS IN CULTURE BY PRESERVING THEIR IMMATURE STATE

It has been a long-standing goal to gain a better understanding of the genes that regulate the self-renewal of HSCs in order to develop optimal culture conditions in which HSCs can be expanded for clinical benefit. In this study, we asked whether inhibition of Lysine-specific histone demethylase 1A (LSD1) would improve expansion of cultured human HSCs derived from umbilical cord blood (UCB). LSD1 modulates gene expression by eliminating di- and mono-methyl groups on H3 lysine K4 and K9 residues and has been shown to negatively impact self-renewal of mouse HSCs. To evaluate the effect of LSD1 inhibition we treated UCB CD34+ cells with three different LSD1 inhibitors (2-PCPA, GSK-LSD1 and RN1) and expanded the cultures for 6 days. Remarkably, treatment with either 2-PCPA or GSK-LSD1 resulted in a more than 10-fold increase of the HSC enriched CD34+EPCR+ population compared to controls. Using CFSE labeling, we found that 2-PCPA did not significantly alter the cell division rate of the cultured CD34+ cells and that the expansion of CD34+EPCR+ cells is not due to increased proliferation, but rather reduced differentiation of the immature HSPCs. Further, we mapped the early transcriptional changes triggered by 2-PCPA in HSCs and found that gene sets corresponding to UCB and fetal liver HSCs were significantly enriched upon 2-PCPA treatment. This suggests that 2-PCPA indeed restricts differentiation and preserves the HSC state upon ex vivo culture. Finally, the frequency of functional HSCs in DMSO and 2-PCPA treated cultures were measured using limiting dilution analysis (LDA). Cultures treated with 2-PCPA showed a 5-fold higher content of long-term repopulating cells compared to DMSO treated controls (1 in 615 vs 1 in 3041, p=0.03). Altogether our data suggest that LSD1 inhibition supports both phenotypic and functional HSCs in culture by preserving the immature state.

The non-homologous end-joining activity is required for Fanconi anemia fetal HSC maintenance

BackgroundRecent studies have shown that deficiency in the Fanconi anemia (FA) DNA repair pathway enhances the error-prone non-homologous end-joining (NHEJ) repair, leading to increased genomic instability, and that genetic or pharmacological inhibition of the NHEJ pathway could rescue the FA phenotype.MethodsFirst, we exposed LSK cells from WT and Fanca−/− mice to DNA-PKcs inhibitor NU7026 or Ku70 knockdown to examine whether inhibition of NHEJ sensitizes Fanca−/− HSPCs to PARP inhibitor (PARPi)- or interstrand crosslinking (ICL)-induced cell death and genomic instability. We then generated DNA-PKcs3A/3AFanca−/− mice to investigate the effect of specific inactivation of NHEJ on fetal HSCs. Lastly, we used two p53 mutant models to test whether specific inactivation of the p53 function in apoptosis is sufficient to rescue embryonic lethality and fetal HSC depletion in Fanca−/−DNA-PKcs3A/3A mice.ResultsInhibition of NHEJ sensitizes HSPCs from Fanca−/− mice to PARP inhibition- and ICL-induced cell death and genomic instability and further decreases Fanca−/− HSPC proliferation and hematopoietic repopulation in irradiated transplant recipients. Specific inactivation of NHEJ activity by the knockin DNA-PKcs3A/3A mutation in two FA mouse models, Fanca−/− and Fancc−/−, leads to embryonic lethality. DNA-PKcs3A/3A causes fetal HSC depletion in developing Fanca−/− embryos due to increased HSC apoptosis and cycling. Both p53−/− and a knockin p53515C mutation, which selectively impairs the p53 function in apoptosis, can rescue embryonic lethality and fetal HSC depletion in Fanca−/−DNA-PKcs3A/3A mice.ConclusionThese results demonstrate that the NHEJ pathway functions to maintain Fanconi anemia fetal HSCs.

Generation of <i>foxn1</i>/ <i>Casper</i> Mutant Zebrafish for Allograft and Xenograft of Normal and Malignant Cells

Cell transplantation into immunodeficient recipients is a widely-used approach to study stem cell and cancer biology; however, studying cell states post transplantation in vivo is inconvenient in mammals. Here, we generated a zebrafish foxn1/Casper mutant that is transparent and immunodeficient with deficient T cells. By employing the mutant for hematopoietic stem cell transplantation (HSCT), we could achieve nonconditioned transplantation. Meanwhile, we found that fetal HSCs from 3 days post fertilization produce a better transplant outcome in adult recipients, compared to adult HSCs. Furthermore, adult HSCs from the newly generated Tg (runx1:en-GFP) line have higher engraftment efficiency than that from the Tg (CD41:GFP) line. In addition to HSCT, foxn1/Casper mutant is feasible to allogeneic leukemia and muscle cell transplantation, as well as xenogenic medaka HSCT. Collectively, foxn1/Casper mutant permits the nonconditioned engraftment of multiple cell types with superiority in imaging and characterization of transplanted cells at single cell resolution in vivo.

Distinct Regulatory Networks Govern Human Hematopoietic Stem Cell Across Development

Introduction: Much of our fundamental understanding of stem cell biology comes from studies of hematopoiesis where single cells produce differentiated progeny while still retaining the ability to produce daughter stem cells (self-renewal). The cardinal property of a stem cell, whether normal or malignant, is self-renewal; the key biological process that ensures the ability of the stem cell to maintain long-term clonal growth. However, our understanding of the molecular basis of self-renewal in human hematopoiesis is limited.At the embryonic stage fetal liver is the main source of hematopoiesis; from week 6 of gestation until before birth. At this stage HSCs are in a different microenvironment but capable of self-renewing and differentiation to the full spectrum of blood lineages. While murine studies uncovered several intrinsic differences between fetal and adult HSCs, a comprehensive analysis of human HSC compartment across development is lacking.In this study we have combined HSC purification methods and xenograft quantitative assay in conjunction with low input RNA sequencing and Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) to provide a comprehensive functional and molecular outlook of human stem cell compartment across development.Results: We followed the dynamics of four sub-fractions of CD34+CD38- divided by CD90 and CD49f expression across human blood development: fetal liver (hFL) and adult bone marrow (hBM). Using xenograft model, we identified human long, intermediate and short term HSCs in hFL and hBM. 5 single CD90+CD49f+ hFL cells were capable of sustaining the multilineage graft for over 52 weeks up to tertiary recipient, while BM cells only last for 20 weeks in the primary recipient. The frequency of LT-HSC in the CD90+CD49f+ compartment goes from 1/8 in hFL to 1/50 in hBM. hFL CD90-CD49f+ cells showed an intermediate repopulation capacity up to 44 weeks in secondary recipient.On average 10% of hFL long term HSC (LT-HSC) were in S/G2/M phase, in contrast only 0.4% of BM LT-HSC were in S/G2/M phase indicating that hFL HSCs are 20 times more in cycle compare to BM.We found that 320 genes were expressed differentially between LT-HSC and multipotent progenitors (MPP) in hBM as oppose to only 32 genes found to be differentially expressed in hFL (FDR<0.1). Interestingly, we found only 2 genes in common between these two groups.ERRBS showed an overall increase in methylation of HSC compartment in hBM compare to hFL and gradual demethylation of lineage associated genes in MPP.Conclusion: Our data indicate that there are distinct regulatory networks that govern hFL and hBM HSC self-renewal. We found very little differences in gene expression between all hFL HCS compartments (average 20 genes) compare to hBM (average 224), indicating that by adulthood self-renewal is becoming more restricted to the LT-HSC compartment. DisclosuresNo relevant conflicts of interest to declare.

A quantitative assessment of the content of hematopoietic stem cells in mouse and human endosteal-bone marrow: a simple and rapid method for the isolation of mouse central bone marrow.

BackgroundIsolation of bone marrow cells, including hematopoietic stem cells, is a commonly used technique in both the research and clinical settings. A quantitative and qualitative assessment of cell populations isolated from mouse and human bone marrow was undertaken with a focus on the distribution of hematopoietic cells between the central bone marrow (cBM) and endosteal bone marrow (eBM).MethodsTwo approaches to cBM isolation from the hind legs were compared using the C57BL/6J and BALB/cJ strains of laboratory mice. The content of hematopoietic stem cells in eBM was compared to cBM from mice and human fetal bone marrow using flow cytometry. Enzymatic digestion was used to isolate eBM and its effects on antigen expression was evaluated using flow cytometry. Humanized immunodeficient mice were used to evaluate the engraftment of human precursors in the cBM and eBM and the effects of in vivo maturation on the fetal stem cell phenotype were determined.ResultsThe two methods of mouse cBM isolation yielded similar numbers of cells from the femur, but the faster single-cut method recovered more cells from the tibia. Isolation of eBM increased the yield of mouse and human stem cells. Enzymatic digestion used to isolate eBM did, however, have a detrimental effect on detecting the expression of the human HSC-antigens CD4, CD90 and CD93, whereas CD34, CD38, CD133 and HLA-DR were unaffected. Human fetal HSCs were capable of engrafting the eBM of immunodeficient mice and their pattern of CD13, CD33 and HLA-DR expression partially changed to an adult pattern of expression about 1 year after transplantation.ConclusionsA simple, rapid and efficient method for the isolation of cBM from the femora and tibiae of mice is detailed. Harvest of tibial cBM yielded about half as many cells as from the femora, representing 6.4 % and 13 %, respectively, of the total cBM of a mouse based on our analysis and a review of the literature. HSC populations were enriched within the eBM and the yield of HSCs from the mouse and human long bones was increased notably by harvest of eBM.Electronic supplementary materialThe online version of this article (doi:10.1186/s12878-015-0031-7) contains supplementary material, which is available to authorized users.

Fev Expression Is Functionally Required for Fetal HSCs and LSCs Indicating Prenatal Origin of Leukemia

Cancers, particularly in the childhood and adolescence, can originate prenatally or postnatally. How to distinguish however and what clinical implication of the distinct origin of cancers remains unknown. Here we have identified and characterized transcription factor fev an indicator of prenatal origin of leukemia in the human context of normal and leukemic hematopoiesis. Fev is expressed in fetal and neonatal and not in adult hematopoietic cells. Functional assays showed fev is essential for self-renewal in fetal HSCs. More interestingly, fev is expressed in leukemic cells of some cell lines and patients, which is associated with more frequent relapse. Fev-knockdown in leukemic cells markedly compromised their capacity of leukemic reconstitution in xenograft recipients. Leukemogenic targeting experiments by forced expression of MLL-AF9 or N-Myc showed fev-regulated self-renewal is retained from fetal HSCs rather than reactivated in adult HSCs. Therefore, fev-expression can distinguish LSCs from normal HSCs and indicate its prenatal origin. DisclosuresNo relevant conflicts of interest to declare.

Nfix Is Required for Hematopoietic Stem- and Progenitor Cell in Vivo Repopulating Potential.

Abstract 2320Hematopoietic stem cells (HSCs) are both necessary and sufficient to sustain the complete blood system of vertebrates. Specified at several locations during fetal development, they ultimately congregate in the fetal liver for rapid expansion. Around birth HSCs relocate to the bone marrow (BM) and enter a state of cellular quiescence, cycling intermittently to supply progenitor cells, which differentiate into the distinct blood lineages. Due to their regenerative potential, HSCs are heavily utilized in the clinic for bone marrow transplants (BMTs) to treat a variety of diseases. However, a lack of suitable donors and inefficiency in recipient engraftment currently limit this life saving therapy. To improve BMT regimens, a better understanding of regulators of HSC BM engraftment is required.Recently, we examined the gene expression patterns of HSCs as they emerge throughout murine ontogeny (McKinney-Freeman et al., Cell Stem Cell, in press). We observed that the transcription factor Nfix, a member of the nuclear factor I (NFI) family of transcription factors never before linked to HSC biology, was highly expressed by both fetal liver and BM HSCs. These data suggest that Nfix may play a novel role in regulating HSC function in the BM and/or fetal liver. To test this hypothesis, HSCs were enriched from E14.5 fetal liver or adult BM (Lineage-, c-kit+, Sca-1+ (LSK) cells) and then transduced with lentiviruses carrying shRNAs targeting Nfix. Twenty-four hours post-transduction, cells were injected into lethally irradiated mice along with untransduced BM LSK competitor cells congenic at the CD45 allele. The peripheral blood of recipient mice was then analyzed periodically over 16 weeks for engraftment of the Nfix-depleted cells. Depletion of Nfix by two independent shRNA (confirmed by Western blot analysis to deplete NFIX protein levels to <20% of baseline) resulted in a significant decrease in the repopulating activity of BM LSK cells relative to LSK cells transduced with either of two independent control shRNAs. As early as two weeks post-transplant, a 22% +/− 5% (p=0.03) reduction in repopulating activity was observed. By 16 weeks post-transplant, this reduction in repopulating potential had gradually increased to 55% +/−8% (p<0.0001) in four independent experiments. Depletion of Nfix in fetal liver-derived LSK cells resulted in a similar loss of repopulating potential. Critically, in vitro analysis of BM LSK expansion in liquid culture and differentiative potential, as analyzed by the methylcellulose based colony forming assay, revealed no differences in the activity of LSK cells transduced with Nfix-specific shRNAs compared to controls. Thus, it is unlikely that the observed decrease in BM repopulating activity is due to general cytotoxicity resulting from Nfix depletion or a block in differentiation. Concordantly, lineage analysis of peripheral blood of recipients showed no significant differences in the percentage of the major blood lineages derived from LSK cells transduced with Nfix-specific shRNAs compared to controls. Thus, the observed decrease in repopulating activity likely occurs at the level of HSCs and multipotent progenitors. In agreement with this conclusion, when BM of recipients transplanted with Nfix-depleted LSK cells are examined 4 and 16 weeks post transplant, a loss of phenotypic HSCs (LSK/CD150+/CD48-) relative to controls is evident.The loss of repopulating potential by Nfix-depleted cells as early as two weeks post-transplant suggests that Nfix may be involved in either the homing/lodgment of HSCs in the BM or their ability to expand soon after incorporation into the stem cell niche. We are presently teasing out the molecular mechanism behind this phenomenon. Furthermore, to examine a role for Nfix during HSC homeostasis, we are currently analyzing mice in which Nfix has been conditionally ablated in the hematopoietic compartment. Finally, functional analysis of two other members of the NFI gene family shown by our array analysis to be expressed by BM HSC, Nfia and Nfic, will further assess a role for this gene family in the regulation of HSCs.In summary, we have for the first time established a role for a member of the NFI gene family, Nfix, in HSC biology, as evident by a decrease in BM repopulating activity in Nfix-depleted HSCs. By dissecting the precise role of Nfix in HSC biology, we will glean insights that could improve our understanding of graft failure in clinical BMTs. Disclosures:No relevant conflicts of interest to declare.

GPI80 Distinguishes Transplantable Human Fetal Hematopoietic Stem Cells From Multipotential Progenitors

Abstract 1280Hematopoietic stem cells (HSCs) are responsible for the lifelong maintenance of all the cells of the blood system. Self-renewal and differentiation of HSCs is tightly regulated by the complex interplay of extrinsic and intrinsic factors that work together to maintain a homeostatic balance. However, the regulatory mechanisms necessary to maintain the stemness of human HSCs are not well understood, largely due to lack of specific surface markers to purify HSCs. In order to facilitate a detailed study of the human long-term HSC and its niche environment, we performed genome-wide gene expression analysis comparing undifferentiated CD34+CD38−CD90+ human fetal hematopoietic stem and progenitor cells (HSPCs) and their progeny (CD34+CD38−CD90−) and attempted to identify novel markers specific to the long-term HSC. Expression analysis identified 58 differentially expressed candidate HSC surface markers, including GPI-80 (Vnn2), a GPI-anchored surface protein recently described in neutrophil adherence and migration. Flow cytometry revealed that only a subpopulation of CD34+CD38−CD90+ HSPCs expressed GPI-80. Strikingly, GPI-80 expression segregated HSPCs into two functionally distinct populations; NSG mice (NOD/Shi-scid IL2rgamma chain null mice) transplanted with CD34+CD90+GPI-80+ cells (herein noted as GPI-80+HSPCs) displayed robust, multilineage human hematopoietic reconstitution, whereas CD34+CD90+GPI-80− cells (GPI-80−HSPCs) showed little to no hematopoietic engraftment. Furthermore, in vitro assays showed that while both populations were multipotent and generated myeloerythroid and B and T lymphoid cells in culture, only the GPI-80+HSPCs demonstrated the ability to self-renew on mouse mesenchymal stem cell stroma, whereas GPI-80−HSPCs displayed loss of self-renewal and subsequent differentiation. These results define GPI-80 as a novel HSC surface marker that can be used to purify transplantable HSCs during human development. To identify regulatory mechanisms that define the engraftable HSC, we selected differentially expressed genes between GPI-80+ and GPI-80− HSPCs and assessed each set for enrichment of Gene Ontology (GO) categories associated with specific biological processes. There was no difference in genes regulating lineage differentiation, consistent with the finding that both populations are multipotential. However, GPI-80+HSPCs were enriched for genes related to cell migration, while the GPI-80− progenitors were enriched for active cell cycle genes. This indicates that GPI-80 may mark a more pure population of quiescent human fetal HSCs that possess unique properties necessary for engraftment. Ongoing studies are focusing on investigating the role of GPI-80 in the homing and trafficking of fetal HSCs, and defining the mechanisms that facilitate interactions between the GPI-80+ HSC and its niche. Disclosures:Magnusson:Novogenix Laboratories LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Hemogenic Endothelium

AbstractAbstract SCI-44Hematopoietic stem cells (HSCs) develop from endothelium in the midgestation mouse conceptus, starting at 10.5 days post coitus, through the formation of intra-aortic clusters. Studies in mouse, zebrafish, chick, and frog embryos established that Runx1 (AML1) is the earliest specific marker of the endothelium from which HSCs and definitive hematopoietic progenitors form. Conditional deletion of Runx1 in vascular endothelial cadherin (VEC) positive cells blocks the emergence of intra-aortic hematopoietic clusters, definitive hematopoietic progenitors (HPs), and HSCs, demonstrating that Runx1 is required in endothelial cells for HP and HSC formation. On the other hand, Runx1 deletion in fetal liver HPs and HSCs with Vav-Cre, or deletion in adult HSCs with Mx1-Cre, does not eliminate HP and HSC function, although it does affect homeostasis. These data (and others) demonstrated that the absolute requirement for Runx1 in HP and HSC formation is transient, and over by the time HPs and HSCs colonize the fetal liver. We are further defining the developmental windows during which Runx1, and the gene encoding its non-DNA binding partner CBFβ, are required for HP and HSC specification and commitment. We have taken two complementary approaches. One approach is to delete Runx1 at specific times and assess the effect on HPs. The second approach is to restore CBFβ function at specific times, or in specific cells in otherwise CBFβ deficient embryos and determine whether that rescues HP or HSC formation. From these experiments we can draw several conclusions: 1) Runx1 is absolutely required before the 40 somite pair stage in the AGM region, as deletion before then severely impairs HP formation or activity; 2) CBFβ activity is required before 9.5 dpc for HP and HSC formation; 3) CBFβ activity in endothelium alone is sufficient for HP formation, but insufficient for HSC formation. Together the data indicate that Runx1-CBFβ activity is required over a period of several days during embryogenesis to specific and commit HSCs. Disclosures:No relevant conflicts of interest to declare.

Effects of Allogeneic Hematopoietic Stem Cell Transplantation Plus Thymus Transplantation on Malignant Tumors: Comparison Between Fetal, Newborn, and Adult Mice

We have recently shown that allogeneic intrabone marrow-bone marrow transplantation + adult thymus transplantation (TT) is effective for hosts with malignant tumors. However, since thymic and hematopoietic cell functions differ with age, the most effective age for such intervention needed to be determined. We performed hematopoietic stem cell transplantation (HSCT) using the intrabone marrow method with or without TT from fetal, newborn, and adult B6 mice (H-2(b)) into BALB/c mice (H-2(d)) bearing Meth-A sarcoma (H-2(d)). The mice treated with all types of HSCT + TT showed more pronounced regression and longer survival than those treated with HSCT alone in all age groups. Those treated with HSCT + TT showed increased numbers of CD4(+) and CD8(+) T cells but decreased numbers of Gr-1/Mac-1 myeloid suppressor cells and decreased percentages of FoxP3 cells in CD4(+) T cells, compared with those treated with HSCT alone. In all mice, those treated with fetal liver cell (as fetal HSCs) transplantation + fetal TT or with newborn liver cell (as newborn HSCs) transplantation (NLT) + newborn TT (NTT) showed the most regression, and the latter showed the longest survival. The number of Gr-1/Mac-1 cells was the lowest, whereas the percentage of CD62L(-)CD44(+) effector memory T cells and the production of interferon γ (IFN-γ) were highest in the mice treated with NLT + NTT. These findings indicate that, at any age, HSCT + TT is more effective against cancer than HSCT alone and that NLT + NTT is most effective.

531. Human Fetal Liver HSCs Demonstrate High Transduction Rates and Sustained Transgene Expression Following Implantation

531. Human Fetal Liver HSCs Demonstrate High Transduction Rates and Sustained Transgene Expression Following Implantation

Necdin Regulates Hematopoietic Stem Cell Quiescence and Sensitivity to Genotoxic Stress.

Abstract 379Necdin, a member of MAGE (melanoma antigen) family proteins, is a growth suppressing protein that was first identified in post mitotic neurons. The gene encoding necdin is one of several deleted in individuals with Prader-Willi syndrome, a neurobehavioural disorder associated with an increased risk of myeloid leukemia. It is reported that necdin interacts with p53 and represses p53-mediated apoptosis in neurons, but its role in hematopoiesis is largely unknown. Recently, we defined a critical role of p53 in regulating hematopoietic stem cell quiescence, and identified necdin as a target gene of p53, that is highly expressed in LT-HSCs (Liu Y et al., Cell Stem Cell, 2009). To define the role of necdin in hematopoiesis, we have analyzed the hematopoietic compartment of necdin-null mice. As necdin-null mice die perinatally, we first investigated fetal hematopoiesis and found no alteration in the frequency of fetal liver HSCs, defined as Lin-Sca1+Mac1+CD48-CD150+ within the fetal liver cells. Although necdin-null fetal liver HSCs increase serial replating capability in methylcellulose and maintain stemness in long-term stromal based cultures better than wild type HSCs, necdin-null fetal liver HSCs repopulate lethally irradiated recipient mice similar to wild type HSCs, in primary, secondary, and tertiary serial bone marrow transplantation assays. In addition, necdin-null HSCs show almost comparable repopulating ability as wild type HSCs, after secondary competitive bone marrow transplantation assays. These imply that necdin is dispensable for HSC self renewal. On the other hand, BM-derived necdin-null HSCs show decreased quiescence 4 months after transplantation, and increased proliferation as indicated by in vivo BrdU incorporation assays. Furthermore, recipient mice repopulated with necdin-null HSCs show enhanced sensitivity both to weekly 5-FU administration and to total body irradiation, as manifested by increased mortality. This suggests that the decreased quiescence of necdin-null HSCs leads to their depletion under conditions of genotoxic stress. Gene expression profiling studies have identified several deregulated signaling pathways in the necdin-null HSCs. Expression of several p53 target genes is altered in irradiated necdin-null HSCs, which may account for their enhanced radiosensitivity. We are now investigating these necdin target genes to clarify how necdin functions to critically regulate HSC quiescence. We are also determining whether targeting necdin could be a therapeutic approach to eliminate quiescent leukemia stem cells, using a murine CML model. Disclosures:No relevant conflicts of interest to declare.

Bcl11b is a positive regulator of T-lineage specification (85.19)

Abstract Hematopoietic stem cells (HSC) commit to the T-lineage by gradually losing developmental plasticity and gaining T cell identity. A recently discovered transcription factor Bcl11b is expected to be a key regulator in this process. It is known Bcl11b is crucial for alpha/beta T cell development and for survival of double positive cells specifically. However, little is known about how Bcl11b works in the very early stages of T cell development. We have cloned cDNA of four transcripts of Bcl11b, and over-expressed these Bcl11b isoforms in fetal liver HSCs. Our data so far has shown that certain Bcl11b isoforms positively regulate T-lineage specification. While full-length Bcl11b and Bcl11b 1-2-4 are growth inhibitory, full-length Bcl11b stimulates emerging of Thy1+ cells from HSCs under a condition that does not normaly favor T cell development. In another experiment, fetal liver HSCs transduced with Bcl11b 1-3-4 and 1-4 were co-cultured with OP9-DL1 cells or OP9 control cells. The data showed that after 4 days of co-culturing HSCs with OP9 control cells, empty vector infected HSCs showed no developmental progression: almost all cells were c-kit+ CD25-; however, some Bcl11b transfected cells became c-kit+, Thy1+ and CD25+ DN2-like cells, normally seen only on OP9-DL1 cells. Our data revealed that certain isoforms of Bcl11b may stimulate T cell development and may even have a novel relationship to Notch signaling.

Mll-AF9 Initiates Fetal Hematopoietic Stem Cell Transformation but Development of Overt Leukemia Requires Additional Events in Post-Natal Cells

Backtracking studies in twins show that human MLL fusion gene leukemias are often initiated during fetal life. However, important unresolved questions arewhat is the nature of additional genetic or epigenetic events that are necessary for development of overt leukemia in addition to the MLL-fusion gene? anddo these additional events occur directly in fetal hematopoietic cells or, alternatively, in the more differentiated progenitor cells that develop post-natally? In order to address these questions, the murine knock-in mouse model which contains the Mll-AF9 oncogene under endogenous regulatory control in all hematopoietic stem and progenitor cells is useful.In the current experiments, we analyzed the characteristics of day E14.5 fetal liver Lin− CD117+ Sca1+ Thy1.1low stem cells (FL-HSCs) and compared them with bone marrow HSCs (BM-HSCs) from 8-wk old mice for transformability and the ability to produce leukemia when transplanted to secondary recipients. Fetal liver cells were stained with biotin labeled antibodies against lineage markers (B220, CD3e, CD4, CD8, Ter119 and Gr1), and then enriched using MACS Micro Beads/Columns (Miltenyi Biotec). Partially enriched Lineage negative (Lin−) cells were further stained with PE-Cy5-Streptavidin, FITC-Sca1, APC-CD117 and PE-Thy1.1 antibodies, and then sorted with a FACS Aria (BD Bioscience). Sorting profiles showed that Mll-AF9 fetal livers had a higher percentage of CD117+ Sca1+ cells in the Lin− population than the fetal livers from wild type fetuses (1.4% vs. 0.9%, P < 0.05), indicating Mll-AF9 fusion gene induced expansion of FL-HSCs by day E14.5. The selfrenewal capability of FL-HSCs was also studied using myeloid colony forming assays in vitro. Sorted HSCs were cultured in methylcellulose medium containing IL-3, IL-6, SCF and GM-CSF, replated every 7 days for three generations and colony numbers were counted for each generation. The study revealed that Mll-AF9 FL-HSCs form significantly higher numbers of myeloid colonies than wild type FL-HSCs in all three generations. Colony distribution analysis also showed that Mll-AF9 FL-HSCs produce more immature compact colonies than wild type FL-HSCs in methylcellulose medium. 94–96% of cultured cells of the third generation were CD11b positive myeloid cells by FACS. The Mll-AF9 induced increase in FL-HSC numbers and colony growth was similar to the results we recently reported (Chen W et al, Cancer Cell , 13, 432, 2008) for BM-HSCs from 8-wk old Mll-AF9 mice. To compare the ability of Mll-AF9 FL-HSCs and BM-HSCs to produce overt leukemia, cells were injected with rescue bone marrow cells into lethally irradiated wild type recipients. One hundred BM-HSCs from 8-wk old Mll-AF9 mice caused leukemia in 90% of recipients at a median of 165 days. On the other hand, one hundred FL-HSCs recipients developed leukemia later and with lower frequency; in the recipients of one hundred FL-HSCs, 60% of recipient mice died at a median of 307 days (p<0.002). Increasing the injected number of FL-HSCs to 1000 did not increase the number of recipients with fatal leukemia when mice were followed to 355 days. Leukemic mice in all groups had enlarged spleens, high white counts and the CD11b/Mac1 myeloid phenotype characteristic of murine Mll-AF9 leukemia. In summary, our results show that in murine cells the Mll-AF9 fusion gene results in transformation both in fetal liver HSCs and BM HSCs as reflected in an increased number of both HSCs and compact myeloid colonies in vitro. However, MLL fusion gene induced transformation in either fetal liver or early postnatal bone marrow, while necessary, is not sufficient to produce overt leukemia in the mouse. The long delay in leukemia development in recipients of fetal HSCs and the similarities in leukemia organ distribution and phenotype in recipients of fetal and post natal HSCs suggests that leukemogenesis is a multistep process with critical secondary events occurring in cells that are post-natal. Therapeutic elimination of these post-natal leukemia-propagating cells in the mouse and the human should be facilitated by further understanding of the molecular signature of these critical cells.