EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes ß-catenin signaling during hematopoietic stem cell development in zebrafish.

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

Lentivirus-Mediated BCL-XL Overexpression Inhibits Stem Cell Apoptosis during Ex Vivo Expansion and Provides Competitive Advantage Following Xenotransplantation.

Hematopoietic reconstitution after hematopoietic stem cell transplantation (HSCT) is influenced by the number of transplanted cells. However, under certain conditions donor cell counts are limited and impair clinical outcome. Hematopoietic stem and progenitor cell (HSPC) expansion prior to HSCT is a widely used method to achieve higher donor cell counts and minimize transplantation-related risks such as graft failure or delayed engraftment. Still, expansion in a non-physiological environment can trigger cell death mechanisms and hence counteract the desired effect. We have shown earlier that during HSCT a relevant amount of HSPCs were lost due to apoptosis and that cell death inhibition in donor HSPCs improved engraftment in xenotransplantation experiments. Here, we assessed the effect of combined ex vivo expansion and cell death inhibition on HSPC yield and their reconstitution potential in vivo. During expansion with cytokines and the small molecule inhibitor StemRegenin 1, concomitant lentiviral overexpression of antiapoptotic BCL-XL resulted in an increased yield of transduced HSPCs. Importantly, BCL-XL overexpression enhanced the reconstitution potential of HSPCs in xenotransplantation experiments in vivo. In contrast, treatment with caspase and necroptosis inhibitors had no favorable effects on HSPC yields nor on cell viability. We postulate that overexpression of antiapoptotic BCL-XL, both during ex vivo expansion and transplantation, is a promising approach to improve the outcome of HSCT in situations with limited donor cell numbers. However, such apoptosis inhibition needs to be transient to avoid long-term sequelae like leukemia.

Stem Cells Collection and Mobilization in Adult Autologous/Allogeneic Transplantation: Critical Points and Future Challenges.

Achieving successful hematopoietic stem cell transplantation (HSCT) relies on two fundamental pillars: effective mobilization and efficient collection through apheresis to attain the optimal graft dose. These cornerstones pave the way for enhanced patient outcomes. The primary challenges encountered by the clinical unit and collection facility within a transplant program encompass augmenting mobilization efficiency to optimize the harvest of target cell populations, implementing robust monitoring and predictive strategies for mobilization, streamlining the apheresis procedure to minimize collection duration while ensuring adequate yield, prioritizing patient comfort by reducing the overall collection time, guaranteeing the quality and purity of stem cell products to optimize graft function and transplant success, and facilitating seamless coordination between diverse entities involved in the HSCT process. In this review, we aim to address key questions and provide insights into the critical aspects of mobilizing and collecting hematopoietic stem cells for transplantation purposes.

A simultaneous knockout knockin genome editing strategy in HSPCs potently inhibits CCR5- and CXCR4-tropic HIV-1 infection.

Allogeneic hematopoietic stem and progenitor cell transplant (HSCT) of CCR5 null (CCR5Δ32) cells can be curative for HIV-1-infected patients. However, because allogeneic HSCT poses significant risk, CCR5Δ32 matched bone marrow donors are rare, and CCR5Δ32 transplant does not confer resistance to the CXCR4-tropic virus, it is not a viable option for most patients. We describe a targeted Cas9/AAV6-based genome editing strategy for autologous HSCT resulting in both CCR5- and CXCR4-tropic HIV-1 resistance. Edited human hematopoietic stem and progenitor cells (HSPCs) maintain multi-lineage repopulation capacity in vivo, and edited primary human T cells potently inhibit infection by both CCR5-tropic and CXCR4-tropic HIV-1. Modification rates facilitated complete loss of CCR5-tropic replication and up to a 2,000-fold decrease in CXCR4-tropic replication without CXCR4 locus disruption. This multi-factor editing strategy in HSPCs could provide a broad approach for autologous HSCT as a functional cure for both CCR5-tropic and CXCR4-tropic HIV-1 infections.

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Circadian disturbances by altering the light-dark cycle negatively affects hematopoietic function of bone marrow in mice.

Circadian rhythms in metabolically active tissues are crucial for maintaining physical health. Circadian disturbance (CD) can cause various health issues, such as metabolic abnormalities and immune and cognitive dysfunctions. However, studies on the role of CD in immune cell development and differentiation, as well as the rhythmic expression of the core clock genes and their altered expression under CD, remain unclear. Therefore, we exposed C57bl/6j mice to repeated reversed light-dark cycles for 90 days to research the effects of CD on bone marrow (BM) hematopoietic function. We also researched the effects of CD on endogenous circadian rhythms, temporally dependent expression in peripheral blood and myeloid leukocytes, environmental homeostasis within BM, and circadian oscillations of hematopoietic-extrinsic cues. Our results confirmed that when the light and dark cycles around mice were frequently reversed, the circadian rhythmic expression of the two main circadian rhythm markers, the hypothalamic clock gene, and serum melatonin, was disturbed, indicating that the body was in a state of endogenous CD. Furthermore, CD altered the temporally dependent expression of peripheral blood and BM leukocytes and destroyed environmental homeostasis within the BM as well as circadian oscillations of hematopoietic-extrinsic cues, which may negatively affect BM hematopoiesis in mice. Collectively, these results demonstrate that circadian rhythms are vital for maintaining health and suggest that the association between CD and hematopoietic dysfunction warrants further investigation.

Age-related Morphofunctional Changes in Sickle Cell Mice Bone Marrow Mesenchymal Stromal Cells.

BACKGROUND AND OBJECTIVES: Bone marrow mesenchymal stromal cells (BM-MSCs) are key elements of the hematopoietic niche and participate in the regulatory mechanisms of hematopoietic stem cells (HSCs). Hematological diseases can affect MSCs and their functions. However, the dysregulations caused by sickle cell disease (SCD) are not fully elucidated. This work explored changes in BM-MSCs and their relationship with age using sickle cell mice (Townes-SS). MATERIALS AND METHODS: BM-MSCs were isolated from Townes-SS, and control groups 30- and 60-day-old Townes-AA and C57BL/6 J. RESULTS: The BM-MSCs showed no morphological differences in culture and demonstrated a murine MSC-like immunophenotypic profile (Sca-1+, CD29+, CD44+, CD90.2+, CD31-, CD45-, and CD117-). Subsequently, all BM-MSCs were able to differentiate into adipocytes and osteocytes in vitro. Finally, 30-day-old BM-MSCs of Townes-SS showed higher expression of genes related to the maintenance of HSCs (Cxcl12, Vegfa, and Angpt1) and lower expression of pro-inflammatory genes (Tnfa and Il-6). However, 60-day-old BM-MSCs of Townes-SS started to show expression of genes related to reduced HSC maintenance and increased expression of pro-inflammatory genes. CONCLUSION: These results indicates age as a modifying factor of gene expression of BM-MSCs in the context of SCD.

RIO-kinase 2 is essential for hematopoiesis.

Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.

ETV2 induces endothelial, but not hematopoietic, lineage specification in birds.

Cardiovascular system develops from the lateral plate mesoderm. Its three primary cell lineages (hematopoietic, endothelial, and muscular) are specified by the sequential actions of conserved transcriptional factors. ETV2, a master regulator of mammalian hemangioblast development, however, is absent in the chicken genome and acts downstream of NPAS4L in zebrafish. Here, we investigated the epistatic relationship between NPAS4L and ETV2 in avian hemangioblast development. We showed that ETV2 is deleted in all 363 avian genomes analyzed. Mouse ETV2 induced LMO2, but not NPAS4L or SCL, expression in chicken mesoderm. Squamate (lizards, geckos, and snakes) genomes contain both NPAS4L and ETV2 In Madagascar ground gecko, both genes were expressed in developing hemangioblasts. Gecko ETV2 induced only LMO2 in chicken mesoderm. We propose that both NPAS4L and ETV2 were present in ancestral amniote, with ETV2 acting downstream of NPAS4L in endothelial lineage specification. ETV2 may have acted as a pioneer factor by promoting chromatin accessibility of endothelial-specific genes and, in parallel with NPAS4L loss in ancestral mammals, has gained similar function in regulating blood-specific genes.

Aging-induced MCPH1 translocation activates necroptosis and impairs hematopoietic stem cell function.

DNA damage contributes to the aging of hematopoietic stem cells (HSCs), yet the underlying molecular mechanisms are not fully understood. In this study, we identified a heterogeneous functional role of microcephalin (MCPH1) in the nucleus and cytoplasm of mouse HSCs. In the nucleus, MCPH1 maintains genomic stability, whereas in the cytoplasm, it prevents necroptosis by binding with p-RIPK3. Aging triggers MCPH1 translocation from cytosol to nucleus, reducing its cytoplasmic retention and leading to the activation of necroptosis and deterioration of HSC function. Mechanistically, we found that KAT7-mediated lysine acetylation within the NLS motif of MCPH1 in response to DNA damage facilitates its nuclear translocation. Targeted mutation of these lysines inhibits MCPH1 translocation and, consequently, compromises necroptosis. The dysfunction of necroptosis signaling, in turn, improves the function of aged HSCs. In summary, our findings demonstrate that DNA damage-induced redistribution of MCPH1 promotes HSC aging and could have broader implications for aging and aging-related diseases.

Single-cell transcriptomics dissects the transcriptome alterations of hematopoietic stem cells in myelodysplastic neoplasms.

BACKGROUND: Myelodysplastic neoplasms (MDS) are myeloid neoplasms characterized by disordered differentiation of hematopoietic stem cells and a predisposition to acute myeloid leukemia (AML). The underline pathogenesis remains unclear. METHODS: In this study, the trajectory of differentiation and mechanisms of leukemic transformation were explored through bioinformatics analysis of single-cell RNA-Seq data from hematopoietic stem and progenitor cells (HSPCs) in MDS patients. RESULTS: Among the HSPC clusters, the proportion of common myeloid progenitor (CMP) was the main cell cluster in the patients with excess blasts (EB)/ secondary AML. Cell cycle analysis indicated the CMP of MDS patients were in an active proliferative state. The genes involved in the cell proliferation, such as MAML3 and PLCB1, were up-regulated in MDS CMP. Further validation analysis indicated that the expression levels of MAML3 and PLCB1 in patients with MDS-EB were significantly higher than those without EB. Patients with high expression of PLCB1 had a higher risk of transformation to AML. PLCB1 inhibitor can suppress proliferation, induce cell cycle arrest, and activate apoptosis of leukemic cells in vitro. CONCLUSION: This study revealed the transcriptomic change of HSPCs in MDS patients along the pseudotime and indicated that PLCB1 plays a key role in the transformation of MDS into leukemia.

A germline point mutation in the MYC-FBW7 phosphodegron initiates hematopoietic malignancies.

Oncogenic activation of MYC in cancers predominantly involves increased transcription rather than coding region mutations. However, MYC-dependent lymphomas frequently acquire point mutations in the MYC phosphodegron, including at threonine 58 (T58), where phosphorylation permits binding via the FBW7 ubiquitin ligase triggering MYC degradation. To understand how T58 phosphorylation functions in normal cell physiology, we introduced an alanine mutation at T58 (T58A) into the endogenous c-Myc locus in the mouse germline. While MYC-T58A mice develop normally, lymphomas and myeloid leukemias emerge in ∼60% of adult homozygous T58A mice. We found that primitive hematopoietic progenitor cells from MYC-T58A mice exhibit aberrant self-renewal normally associated with hematopoietic stem cells (HSCs) and up-regulate a subset of MYC target genes important in maintaining stem/progenitor cell balance. In lymphocytes, genomic occupancy by MYC-T58A was increased at all promoters compared with WT MYC, while genes differentially expressed in a T58A-dependent manner were significantly more proximal to MYC-bound enhancers. MYC-T58A lymphocyte progenitors exhibited metabolic alterations and decreased activation of inflammatory and apoptotic pathways. Our data demonstrate that a single point mutation stabilizing MYC is sufficient to skew target gene expression, producing a profound gain of function in multipotential hematopoietic progenitors associated with self-renewal and initiation of lymphomas and leukemias.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Monoallelic KRAS (G13C) mutation triggers dysregulated expansion in induced pluripotent stem cell-derived hematopoietic progenitor cells.

BACKGROUND: Although oncogenic RAS mutants are thought to exert mutagenic effects upon blood cells, it remains uncertain how a single oncogenic RAS impacts non-transformed multipotent hematopoietic stem or progenitor cells (HPCs). Such potential pre-malignant status may characterize HPCs in patients with RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). This study sought to elucidate the biological and molecular alterations in human HPCs carrying monoallelic mutant KRAS (G13C) with no other oncogene mutations. METHODS: We utilized induced pluripotent stem cells (iPSCs) derived from two unrelated RALD patients. Isogenic HPC pairs harboring either wild-type KRAS or monoallelic KRAS (G13C) alone obtained following differentiation enabled reliable comparative analyses. The compound screening was conducted with an established platform using KRAS (G13C) iPSCs and differentiated HPCs. RESULTS: Cell culture assays revealed that monoallelic KRAS (G13C) impacted both myeloid differentiation and expansion characteristics of iPSC-derived HPCs. Comprehensive RNA-sequencing analysis depicted close clustering of HPC samples within the isogenic group, warranting that comparative studies should be performed within the same genetic background. When compared with no stimulation, iPSC-derived KRAS (G13C)-HPCs showed marked similarity with the wild-type isogenic control in transcriptomic profiles. After stimulation with cytokines, however, KRAS (G13C)-HPCs exhibited obvious aberrant cell-cycle and apoptosis responses, compatible with "dysregulated expansion," demonstrated by molecular and biological assessment. Increased BCL-xL expression was identified amongst other molecular changes unique to mutant HPCs. With screening platforms established for therapeutic intervention, we observed selective activity against KRAS (G13C)-HPC expansion in several candidate compounds, most notably in a MEK- and a BCL-2/BCL-xL-inhibitor. These two compounds demonstrated selective inhibitory effects on KRAS (G13C)-HPCs even with primary patient samples when combined. CONCLUSIONS: Our findings indicate that a monoallelic oncogenic KRAS can confer dysregulated expansion characteristics to non-transformed HPCs, which may constitute a pathological condition in RALD hematopoiesis. The use of iPSC-based screening platforms will lead to discovering treatments that enable selective inhibition of RAS-mutated HPC clones.

The difficult translational pathway from animal models to patients.

Lee et al.1 analyzed the impacts of lentiviral vector transduction and CRISPR-Cas9/homology-directed repair editing on hematopoietic stem and progenitor cell (HSPC) engraftment and clonal dynamics. The study suggests that relative to lentiviral-vector-mediated gene addition, homology-directed repair editing is inefficient in vivo and might impair the engraftment and differentiation of HSPCs.

Genetic haute couture to block HIV-1 at front doors.

The chemokine receptors CCR5 and CXCR4 are "front doors" for HIV-1 infection in host cells, and their targeting represents a potential solution for a cure. Dudek et al.1 now propose a new gene editing strategy to simultaneously block CCR5- and CXCR4-mediated HIV-1 entry in autologous hematopoietic stem and progenitor cells (HSPCs).

Ex vivo expansion potential of murine hematopoietic stem cells is a rare property only partially predicted by phenotype.

The scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCs ex vivo. After 3 weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential for ex vivo self-renewal, with alternative outcomes revealed by single-cell multimodal RNA and ATAC sequencing profiling. While most HSC progeny offered only transient in vivo reconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations for ex vivo HSC expansion, with major implications also for assessment of normal HSC activity.

Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.