Fetal Liver Hematopoietic Stem Research Articles

Beclin 1 prevents ISG15-mediated cytokine storms to secure fetal hematopoiesis and survival.

Proper control of inflammatory responses is essential for embryonic development, but the underlying mechanism is poorly understood. Here, we show that under physiological conditions, inactivation of ISG15, an inflammation amplifier, is associated with the interaction of Beclin 1 (Becn1), via its evolutionarily conserved domain, with STAT3 in the major fetal hematopoietic organ of mice. Conditional loss of Becn1 caused sequential dysfunction and exhaustion of fetal liver hematopoietic stem cells, leading to lethal inflammatory cell-biased hematopoiesis in the fetus. Molecularly, the absence of Becn1 resulted in the release of STAT3 from Becn1 tethering and subsequent phosphorylation and translocation to the nucleus, which in turn directly activated the transcription of ISG15 in fetal liver hematopoietic cells, coupled with increased ISGylation and production of inflammatory cytokines, whereas inactivating STAT3 reduced ISG15 transcription and inflammation but improved hematopoiesis potential, and further silencing ISG15 mitigated the above collapse in the Becn1-null hematopoietic lineage. The Becn1/STAT3/ISG15 axis remains functional in autophagy-disrupted fetal hematopoietic organs. These results suggest that Becn1, in an autophagy-independent manner, secures hematopoiesis and survival of the fetus by directly inhibiting STAT3/ISG15 activation to prevent cytokine storms. Our findings highlight a previously undocumented role of Becn1 in governing ISG15 to safeguard the fetus.

Path of differentiation defines human macrophage identity.

Macrophages play central roles in immunity, wound healing, and homeostasis - a functional diversity that is underpinned by varying developmental origins. The impact of ontogeny on properties of human macrophages is inadequately understood. We demonstrate that definitive human fetal liver (HFL) hematopoietic stem cells (HSCs) possess two divergent paths of macrophage specification that lead to distinct identities. The monocyte-dependent pathway exists in both prenatal and postnatal hematopoiesis and generates macrophages with adult-like responses properties. We now uncover a fetal-specific pathway of expedited differentiation that generates tissue resident-like macrophages (TRMs) that retain HSC-like self-renewal programs governed by the aryl hydrocarbon receptor (AHR). We show that AHR antagonism promotes TRM expansion and mitigates inflammation in models of atopic dermatitis (AD). Overall, we directly connect path of differentiation with functional properties of macrophages and identify an approach to promote selective expansion of TRMs with direct relevance to inflammation and diseases of macrophage dysfunction.

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.

Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia

Demand-adjusted and cell type specific rates of protein synthesis represent an important safeguard for fate and function of long-term hematopoietic stem cells. Here, we identify increased protein synthesis rates in the fetal hematopoietic stem cell pool at the onset of hematopoietic failure in Fanconi Anemia, a prototypical DNA repair disorder that manifests with bone marrow failure. Mechanistically, the accumulation of misfolded proteins in Fancd2−/− fetal liver hematopoietic stem cells converges on endoplasmic reticulum stress, which in turn constrains midgestational expansion. Restoration of protein folding by the chemical chaperone tauroursodeoxycholic acid, a hydrophilic bile salt, prevents accumulation of unfolded proteins and rescues Fancd2−/− fetal liver long-term hematopoietic stem cell numbers. We find that proteostasis deregulation itself is driven by excess sterile inflammatory activity in hematopoietic and stromal cells within the fetal liver, and dampened Type I interferon signaling similarly restores fetal Fancd2−/− long-term hematopoietic stem cells to wild type-equivalent numbers. Our study reveals the origin and pathophysiological trigger that gives rise to Fanconi anemia hematopoietic stem cell pool deficits. More broadly, we show that fetal protein homeostasis serves as a physiological rheostat for hematopoietic stem cell fate and function.

Genetic and Pharmacological Interruption of the SIX1/EYA2 Axis Impairs Leukemia Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

FLT3-Ligand Alone Enables the Identification and Maintenance in Vitro of Viable Quiescent Human Fetal Liver Hematopoietic Stem Cells with Maintenance of Their Function

Cells with sustained multi-lineage blood cell regenerative potential are referred to as hematopoietic stem cells (HSCs). Current findings indicate that they are responsible for lifelong blood production, are first detected within the first month of development in humans and are largely created prior to birth after which they expand their numbers in response to physiological demands for normal or enhanced blood cell output requirements. Interestingly, a number of the intrinsically determined functional properties of HSCs also change after birth. These include an apparent decline in their self-renewal potential. Thus, for example, in humans as in mice, cells with long-term (>6 month) HSC regenerative properties present in the developing fetal liver (FL) are a source of HSCs that possess a much higher in vivo regenerative capability than those from older donor sources, such as adult bone marrow or even cord blood (CB). Accordingly, it has been of longstanding interest to better understand the molecular regulation of this high regenerative capacity prevalent in fetal HSCs for potential future therapeutic as well as scientific exploitation. Here, we describe the results of experiments designed to answer the hypothesis that human FL HSCs with human-relevant self-renewal properties can be isolated as a quiescent CD49f+ subset of the GPI80+CD90+CD38-CD45RA-CD34+CD45+ population following their incubation in standard serum-free culture medium supplemented with FLT3-ligand (FLT3-L) alone. Initial experiments showed expression of the CD49f integrin on first trimester hFL cells selectively depleted cells able to produce colonies of granulocytes, macrophages or erythroid cells in standard 2-week methylcellulose cultures containing SCF, GM-CSF, IL6, IL3 and EPO. Conversely, expression of the CD49f integrin selectively enriched for cells with 12-week output capabilities in both growth factor (GF)-supplemented stromal co-cultures and in sublethally irradiated, transplanted immunodeficient NOD-Rag1 -/-IL2Rγc -/- W 41/41 (NRG-W) mice. Initial experiments designed to test the effect of multiple GF and small molecule additives on the maintenance over a 7-day period of this in vivo regenerative ability of the input FL HSC confirmed GPI80 expression to be a continuing positive selective phenotype. In addition, the result of transplant experiments showed that a 2-day incubation in FLT3-L alone maintained the 12-week serially transplantable activity of the HSCs (12-weeks/cycle) as fully equivalent to the unmanipulated input cells and significantly superior (P<0.05) to FLT3-L+IL3+SF (3GF) with or without addition of UM171, stemregenin, or eltrombopag. Interestingly, in vitro monitoring of GPI80+ cells showed FLT3-L alone maintained the survival of only 20% of the input GPI80+ cells compared to any of the 3GF-based conditions. In addition, the time to complete a first division and subsequent divisions of the input cells was delayed and prolonged, respectively, in the FLT3-L versus the 3GF conditions. Subsequent 7-day suspension cultures and 6-week GF-supplemented stromal co-cultures experiments have confirmed the existence of a G0 population within the HSC-enriched subset of human FL cells that best maintains their growth potential in FLT3-L alone. Together, these results demonstrate CD49f expression to be a pervasive marker of human HSCs throughout development and reveal the importance of different GF conditions to support the maintenance of viability and retention of self-renewal capability of human fetal HSCs in contrast to those required to activate/support a rapid initiation of cell division. These findings set the stage for future development of strategies to exploit human FL cells therapeutically and may also be critical to designing conditions that will best support the ex vivo maintenance of HSCs at and after birth.

Activating transcription factor 4 in erythroid development and -thalassemia: a powerful regulator with therapeutic potential.

Activating transcription factor 4 (ATF4) is a fundamental basic region/leucine zipper transcription factor, responds to various stress signals, and plays crucial roles in normal metabolic and stress response processes. Although its functions in human health and disease are not completely understood, compelling evidence underscores ATF4 is indispensable for multiple stages and lineages of erythroid development, including the regulation of fetal liver hematopoietic stem cells, induction of terminal erythroid differentiation, and maintenance of erythroid homeostasis. -Thalassemia is a blood disorder arising from mutations in the -globin gene. Reactivating the expression of the -globin gene in adult patients has emerged as a promising therapeutic strategy for ameliorating clinical symptoms associated with -thalassemia. Recent research has suggested that ATF4 contributes to decreased fetal hemoglobin (HbF) level through its binding to potent negative regulators of HbF, such as BCL11A and MYB. Notably, evidence also suggests a contrasting outcome where increased ATF4 protein levels are associated with enhanced HbF at the transcriptional level. Consequently, the identification of mechanisms that modulate ATF4-mediated -globin transcription and its effects on erythroid development may unveil novel targets for -thalassemia treatment.

PTEN regulates hematopoietic lineage plasticity via PU.1-dependent chromatin accessibility

PTEN loss in fetal liver hematopoietic stem cells (HSCs) leads to alterations in myeloid, T-, and B-lineage potentials and T-lineage acute lymphoblastic leukemia (T-ALL) development. To explore the mechanism underlying PTEN-regulated hematopoietic lineage choices, we carry out integrated assay for transposase-accessible chromatin using sequencing (ATAC-seq), single-cell RNA-seq, and invitro culture analyses using invivo-isolated mouse pre-leukemic HSCs and progenitors. We find that PTEN loss alters chromatin accessibility of key lineage transcription factor (TF) binding sites at the prepro-B stage, corresponding to increased myeloid and T-lineage potentials and reduced B-lineage potential. Importantly, we find that PU.1 is an essential TF downstream of PTEN and that altering PU.1 levels can reprogram the chromatin accessibility landscape and myeloid, T-, and B-lineage potentials in Ptennull prepro-B cells. Our study discovers prepro-B as the key developmental stage underlying PTEN-regulated hematopoietic lineage choices and suggests a critical role of PU.1 in modulating the epigenetic state and lineage plasticity of prepro-B progenitors.

Replication stress increases mitochondrial metabolism and mitophagy in FANCD2 deficient fetal liver hematopoietic stem cells.

Fanconi Anemia (FA) is an inherited bone marrow (BM) failure disorder commonly diagnosed during school age. However, in murine models, disrupted function of FA genes leads to a much earlier decline in fetal liver hematopoietic stem cell (FL HSC) number that is associated with increased replication stress (RS). Recent reports have shown mitochondrial metabolism and clearance are essential for long-term BM HSC function. Intriguingly, impaired mitophagy has been reported in FA cells. We hypothesized that RS in FL HSC impacts mitochondrial metabolism to investigate fetal FA pathophysiology. Results show that experimentally induced RS in adult murine BM HSCs evoked a significant increase in mitochondrial metabolism and mitophagy. Reflecting the physiological RS during development in FA, increase mitochondria metabolism and mitophagy were observed in FANCD2-deficient FL HSCs, whereas BM HSCs from adult FANCD2-deficient mice exhibited a significant decrease in mitophagy. These data suggest that RS activates mitochondrial metabolism and mitophagy in HSC.

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.

Clonal Analysis Reveals Functional and Transcriptional Heterogeneity of Self-Renewing Fetal Liver Hematopoietic Stem Cells

The fetal liver (FL) is a key organ supporting the expansion of hematopoietic stem cells (HSCs) during development. The ability to expand transplantable HSCs in vitro is a long-standing challenging; thus, decoding the gene pathways that regulate proliferation and self-renewal during FL-HSC development is highly relevant to engineering HSCs for therapeutic applications. Moreover, while heterogeneity in self-renewal and engraftment properties of HSCs in adult bone marrow has been extensively studied, these properties remain largely uncharacterized in FL-HSCs. To explore FL-HSC properties at the single cell level, we engineered a serum-free culture platform designed to recapitulate the murine FL endothelial niche (FL-EC) (Figure1), and sorted single FL-HSC (CD45+GR1-F4/80-SCA1highEPCRhigh) into individual wells containing FL-EC. Following co-culture, we observed emergence of immunophenotypically distinct types of hematopoietic colonies as determined by flow cytometry. Only colonies containing cells predominantly expressing high levels of HSC markers SCA1 and EPCR (Figure1, HSC colonies) provided multilineage engraftment as determined by transplantation assays, whereas colonies containing cells expressing HSC markers and more differentiated progeny (mixed colonies) were unable to provide serial multilineage engraftment. Furthermore, cellular progeny from a single HSC colony transplanted into multiple recipients demonstrated serial engraftment, indicating the FL-EC niche could amplify single FL-HSC with long-term reconstitution capacity. These studies suggest that transplantable FL-HSCs are uniquely distinguished by their propensity for symmetric self-renewal in this assay. However, not all HSC colonies provided engraftment in secondary recipients, suggesting heterogeneity in self-renewal properties of immunophenotypically defined FL-HSCs that required deeper analysis. Thus, to further assess transcriptional heterogeneity in HSC colonies, we next integrated single-cell RNA sequencing (scRNAseq) into the clonal FL-HSC assay (Figure 2). Residual cells from six HSC colonies were divided for simultaneous scRNAseq and transplantation. Consistent with experiments above, we observed heterogeneity in long-term serial engraftment from individual colonies. Remarkably, cells from the colony capable of serial engraftment were distinctly enriched in genes associated with dormant HSCs in adult marrow (Cabezas-Wallscheid et.al., Cell 2017). Conversely, cells from HSC colonies that lacked serial engraftment capacity exhibited increased expression of Myc target genes by Gene Set Enrichment Analysis (GSEA), and ribosome biogenesis and mitochondrial translation by Gene Ontogeny (GO) analysis, which are hallmarks of biosynthetically activated HSCs in adult marrow that are primed for differentiation to progenitor states. To further assess the transcriptional heterogeneity of primary FL-HSC, we next performed scRNAseq with freshly isolated FL-HSC. Unsupervised clustering and co-regulated modules of differentially expressed genes identified a rare subset of cells highly enriched for genes associated with HSC dormancy, while the other cells featured higher expression of genes involved in biosynthetic processes via GO analysis (e.g. cytoplasmic translation, rRNA processing). This data further supports transcriptional heterogeneity within the FL-HSC compartment, which includes a unique biosynthetically dormant subset. Collectively, our studies elucidate previously unrecognized heterogeneity in FL-HSCs, highlighting a state of relative biosynthetic dormancy as a key to symmetric self-renewal of serially engraftable FL-HSCs. These findings complement recent studies demonstrating distinct developmental origins for multipotent progenitors and functional engraftable HSCs in definitive hematopoiesis (Dignum et.al., Cell Reports 2021; Patel et.al., Nature 2022), suggesting functional heterogeneity in immunophenotypically defined FL-HSCs in our study may in part reflect these distinct ontogenies. Building on these studies, we are currently using CRISPR-Cas9 gene knockout to define the roles of candidate genes identified by our scRNAseq analysis that are implicated in regulating dormancy and symmetric self-renewal of FL-HSCs, which would provide essential insight for future strategies for ex vivo amplification of HSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Isolation and Characterization of Fetal Liver Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are responsible for the generation and maintenance of pools of multipotent precursors that ultimately give rise to all fully differentiated blood and immune cells. Proper identification and isolation of HSCs for functional analysis has greatly facilitated our understanding of both normal and abnormal adult hematopoiesis. Whereas adult hematopoiesis inmice and humans is driven by quiescent HSCs that reside almost exclusively within the bone marrow (BM), developmental hematopoiesis is characterized by a series of transient progenitors driving waves of increasingly mature hematopoietic cell production that occur across multiple anatomical sites. These waves of hematopoietic cell production are also responsible for the generation of distinct immune cell populations during development that persist into adulthood and contribute uniquely to adult immunity. Therefore, methods to properly isolate and characterize fetal progenitors with high purity across development become increasingly important not only for defining developmental hematopoietic pathways, but also for understanding the contribution of developmental hematopoiesis to the immune system. Here, we describe and discuss methods and considerations for the isolation and characterization of HSCs from the fetal liver, the primary hematopoietic organ during fetal development.

Multi-modal profiling of human fetal liver hematopoietic stem cells reveals the molecular signature of engraftment

The human hematopoietic stem cell harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, hematopoietic stem cells in the fetal liver undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. Here, we profile 26,407 fetal liver cells at both the transcriptional and protein level including ~7,000 highly enriched and functional fetal liver hematopoietic stem cells to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression reveals a generalizable signature defining functional fetal liver hematopoietic stem cells and allows for the stratification of enrichment strategies with high translational potential. More precisely, our integrated analysis identifies CD201 (endothelial protein C receptor (EPCR), encoded by PROCR) as a marker that can specifically enrich for engraftment potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers. As such, it serves as a useful resource for the field and forms the basis for further biological exploration of strategies to retain the engraftment potential of hematopoietic stem cells ex vivo or induce this potential during in vitro hematopoietic stem cell generation.

Spatial transcriptome profiling by MERFISH reveals fetal liver hematopoietic stem cell niche architecture

The hematopoietic stem cell (HSC) niche has been extensively studied in bone marrow, yet a more systematic investigation into the microenvironment regulation of hematopoiesis in fetal liver is necessary. Here we investigate the spatial organization and transcriptional profile of individual cells in both wild type (WT) and Tet2−/− fetal livers, by multiplexed error robust fluorescence in situ hybridization. We find that specific pairs of fetal liver cell types are preferentially positioned next to each other. Ligand-receptor signaling molecule pairs such as Kitl and Kit are enriched in neighboring cell types. The majority of HSCs are in direct contact with endothelial cells (ECs) in both WT and Tet2−/− fetal livers. Loss of Tet2 increases the number of HSCs, and upregulates Wnt and Notch signaling genes in the HSC niche. Two subtypes of ECs, arterial ECs and sinusoidal ECs, and other cell types contribute distinct signaling molecules to the HSC niche. Collectively, this study provides a comprehensive picture and bioinformatic foundation for HSC spatial regulation in fetal liver.

The Hematopoietic Reconstitution through Intra-Bone-Marrow Injection Using Murine Fetal Liver Hematopoietic Stem Cells

The Hematopoietic Reconstitution through Intra-Bone-Marrow Injection Using Murine Fetal Liver Hematopoietic Stem Cells

Taspase1 orchestrates fetal liver hematopoietic stem cell and vertebrae fates by cleaving TFIIA.

Taspase1, a highly conserved threonine protease encoded by TASP1, cleaves nuclear histone-modifying factors and basal transcription regulators to orchestrate diverse transcription programs. Hereditary loss-of-function mutation of TASP1 has recently been reported in humans as resulting in an anomaly complex syndrome, which manifests with hematological, facial, and skeletal abnormalities. Here, we demonstrate that Taspase1-mediated cleavage of TFIIAα-β, rather than of MLL1 or MLL2, in mouse embryos was required for proper fetal liver hematopoiesis and correct segmental identities of the axial skeleton. Homozygous genetic deletion of Taspase1 disrupted embryonic hematopoietic stem cell self-renewal and quiescence states and axial skeleton fates. Strikingly, mice carrying knockin noncleavable mutations of TFIIAα-β, a well-characterized basal transcription factor, displayed more pronounced fetal liver and axial skeleton defects than those with noncleavable MLL1 and MLL2, 2 trithorax group histone H3 trimethyl transferases. Our study offers molecular insights into a syndrome in humans that results from loss of TASP1 and describes an unexpected role of TFIIAα-β cleavage in embryonic cell fate decisions.

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.

Fetal liver hematopoietic stem cells possess the mast cell repopulation ability within a limited time window

Abstract Recent progress in developmental immunology has revealed the hematopoietic stem cell (HSC)-independent ontogeny for the various hematopoietic lineages, including mast cells (MC). Adult bone marrow (BM) HSCs fail to generate MC in both lineage tracing and transplantation assays. However, it is unclear whether fetal liver (FL) HSCs can generate MC in vivo. We investigated the MC potential of HSCs from FL and the aorta-gonad-mesonephros (AGM) in lineage tracing and transplantation assays. We employed different genetic mouse models that allowed us to identify the Tomato+ progeny of hematopoietic precursors (Runx1mer-cre-mer), endothelial cells (EC, Cdh5ERT2Cre) and HSCs (Fgd5ERT2Cre) by injecting tamoxifen (TAM) at different embryonic days starting at E7.5. TdTomato (Tom)+ HSCs and MCs were quantified after birth. In addition, we transplanted HSCs from AGM (11.5), FL (E12.5 and E15.5), and adult BM (>6 wks) into irradiated mice. Results: The EC-labeling at E7.5 efficiently generated Tomato+ MC, but not HSCs (similar results were obtained for the Runx1mer-cre-mer model). EC-labeling at E9.5 made equivalent Tom+ labeling efficiency in MC and HSCs (~85%). However, E11.5 EC mostly failed to mark MC, but still generated Tom+ HSCs (~25%). HSC-labeling at E14 or postnatal day 2 efficiently marked HSCs, but not MC. Collectively, our results indicate that active MC production terminates before E12 and that HSCs after E14 lack MC potential. Our transplantation study also shows lack of MC repopulation by HSCs after E15. However, E11.5 AGM and E12.5 FL HSCs repopulated MC, revealing a transient MC production potential of HSCs. Conclusion: MC production by hemogenic ECs terminates before E12 and early HSCs produce MC within a limited time window.

Lineage tracing and transplantation studies indicate endothelial origin of B-1a cells in the mouse embryo

Abstract Innate-immune like B-1a cells are unique in its functions and developmental origin compared to conventional adaptive immune B-cells (B-2 cells). Especially, their origin and developmental pathway have been argued over four decades between two proposed models: the lineage model and the selection model. Although many transplantation studies support the lineage model in which B-1a cells are derived from fetal progenitors, contradictory results of fetal liver transplantation studies raise a question as to whether fetal liver hematopoietic stem cells (HSCs) are the primary source of the peritoneal B-1a cells. In order to address this question, we took two approaches using transplantation assays and lineage tracing mouse models that enable us to trace Tomato+ progeny of endothelial cells (ECs, Cdh5CreERT mice) and HSCs (Fgd5CreERT2 mice) at a time of Tamoxifen (Tam) injection, respectively. Results: First, we transplanted HSC-precursors (pre-HSCs) from the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic (E) 11.5 into irradiated NSG neonates. Interestingly, we found more B-1a repopulating capacity than multi-lineage in the pre-HSC population. The lineage tracing of hemogenic ECs revealed that B-1a progenitors and HSCs were simultaneously produced from ECs during E9.5 to 10.5, but B-1a production is diminished after E11.5 while HSC were still labeled at E11.5. The HSC-tracing model demonstrated the minimal contribution of FL HSCs to the postnatal B-1a cell pool. Conclusion: Our data addressed the long-lasting controversy and showed HSC-independent B-1a cell development in the mouse embryo.