Long-term Repopulation Research Articles

Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells

AbstractHematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

Liver specification of human iPSC-derived endothelial cells transplanted into mouse liver

Background & AimsLiver sinusoidal endothelial cells (LSECs) are important in liver development, regeneration, and pathophysiology, but the differentiation process underlying their tissue-specific phenotype is poorly understood and difficult to study because primary human cells are scarce. The aim of this study was to use human induced pluripotent stem cell (hiPSC)-derived LSEC-like cells to investigate the differentiation process of LSECs. MethodshiPSC-derived endothelial cells (iECs) were transplanted into the livers of Fah−/−/Rag2−/−/Il2rg−/− mice and assessed over a 12-week period. Lineage tracing, immunofluorescence, flow cytometry, plasma human factor VIII measurement, and bulk and single cell transcriptomic analysis were used to assess the molecular and functional changes that occurred following transplantation. ResultsProgressive and long-term repopulation of the liver vasculature occurred as iECs expanded along the sinusoids between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptomes of transplanted cells between 1 and 12 weeks after transplantation were compared against primary human adult LSECs. This demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1, GATA4, and FOS have a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process included EMCN and CLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contained zonal subpopulations with a region-specific phenotype. ConclusionsCollectively, this study confirms that hiPSCs can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs. Impact and implicationsLiver sinusoidal endothelial cells (LSECs) are important cells for liver biology, but better model systems are required to study them. We present a pluripotent stem cell xenografting model that produces human LSEC-like cells. A detailed and longitudinal transcriptomic analysis of the development of LSEC-like cells is included, which will guide future studies to interrogate LSEC biology and produce LSEC-like cells that could be used for regenerative medicine.

CD90-Targeted Cocal-Pseudotyped Lentivirus As a Robust Platform for Human HSC Gene Therapy

Ex vivo gene delivery and editing in hematopoietic stem and progenitor cells (HSPCs) provide a promising approach for the treatment of many chronic conditions such as hemoglobinopathies, lysosomal storage diseases, and HIV. However, such approaches require highly specialized biomedical infrastructure limiting its accessibility to most patients who could benefit. The development of a targeted in vivo approach to HSPC gene delivery/editing is therefore necessary for the broadened use of gene therapies. The CD34 +CD90 + subset of HSPCs represents a highly enriched population for both short-term and long-term hematopoietic lineage repopulation capacity as seen in the non-human primate (NHP) transplant model. Furthermore, ex vivo gene editing of solely CD34 +CD90 + Human and NHP hematopoietic stem cells (HSCs) was sufficient to produce therapeutic thresholds for sickle cell phenotypic amelioration after transplantation. Thus, the development of novel gene therapy vectors targeting the CD34 +CD90 + HSC population is crucial for more accessible and robust gene therapy platforms. Here we report the development of a novel CD90-targeted Cocal pseudotyped lentivirus system capable of targeted transduction of HSPCs in vitro and in vivo. Additionally, we compare transduction efficiency and specificity of this new technology with the more routinely used VSVG targeting methodology. To produce targeted lentivirus, native targeting of VSVG or Cocal envelope to the LDL-Receptor (LDLR) was knocked out by amino acid substitution. An anti-CD90 single-chain variable fragment (scFv) was introduced onto the mutated capsids using CD8 hinge and ICAM transmembrane domains. Mutagenesis of Cocal envelope LDLR binding motif was performed by Gibson cloning and confirmed via nanopore sequencing. Successful mutated viral production was assessed using hydrodynamic titration methods followed by functional analysis of transduction on CD90 + cell lines. In vitro transduction specificity and efficiency assays were performed on Human GCSF-mobilized CD34 +enriched apheresis samples and analyzed by flow cytometry. Humanized NBSGW mice were treated with targeted-lentiviral particles via intravenous (IV) and intraosseous (IO) injection. Mice were necropsied 4-8 weeks post injection. Transgene expression was analyzed by flow cytometry or luminescence imaging and confirmed by PCR on human-CD34 + FACS-sorted cells from necropsied mice. CD90-Targeted Cocal-pseudotyped lentivirus (Cocal-CD90) transduced CD34 +CD90 + HSCs 2.12-fold (p=.017) more robustly than CD90-targeted VSVG-pseudotyped lentivirus (VSVG-CD90) in vitro. Cocal-CD90 virus also showed significantly greater on-target transduction specificity of CD34 +CD90 + HSCs (p=.0008) compared to untargeted wild-type Cocal-pseudotyped lentivirus (Cocal-WT); but similar specificity to VSVG-CD90 virus (p=.118). Unlike VSVG-CD90 virus, we did not observe decreased transduction efficiency of CD34 +CD90 + HSCs using Cocal-CD90 virus in comparison to Cocal-WT virus. IV and IO injection of both CD90-targeted viruses in humanized mice resulted in transduction of immobilized human HSPCs (CD34 +CD38 -CD133 +) in the bone marrow. Transduced cells maintained multilineage differentiation capacity as seen by transgene expression of T-cell, B-cell, and myeloid compartments in the bone marrow, spleen, and peripheral blood at necropsy. As observed in vitro, Cocal-CD90 treated mice consistently displayed more robust transgene expression than VSVG-CD90 treated mice. Importantly, transduction of off-target organs (e.g., liver and lungs) post-IV injection with targeted virus was not detected by luminescence imaging during 8 weeks of follow-up or at necropsy. We provide the first evidence of targeted engineering of a knockout Cocal envelope using the attenuation of native LDLR tropism coupled with a CD90-scFv fusogen protein. Cocal-CD90 virus has increased specificity for CD90 + transduction compared to Cocal-WT lentivirus, without lowering transduction efficiency of on-target cells. Furthermore, in comparison to targeted VSVG engineering, Cocal engineered virus does not decrease transduction efficiency of HSCs in vitro or in vivo. Coupled with Cocal lentiviruses' resistance to human serum inactivation and low immunogenicity, targeted Cocal viral vectors are more suitable for future in vivo gene delivery platforms than their VSVG counterparts.

Single Cell Analysis Identifies Expression of PD-L2 on Human Bone Marrow Hematopoietic Stem Cells

The identification of specific and reliable markers for human hematopoietic stem cells (HSCs) is crucial to enable their prospective isolation allowing the investigation of unique physiological HSC functions within the blood cell hierarchy. Despite significant advancements in identification of surface markers for human bone marrow HSC isolation, the enriched populations remain heterogeneous. Here we applied single cell CITE-Seq (cellular indexing of transcriptomes and epitopes) of FACS-enriched hematopoietic stem and progenitor cells (HSPCs) to decipher differentiation hierarchies and to identify markers of most immature populations. We FACS-isolated over 60.000 high-quality human bone marrow HSPCs by sorting CD34 + and CD34 +CD38 - cells from 15 different healthy donors. These were subjected to scCITE-Seq using the BD Rhapsody system with a targeted gene panel comprising 600 most relevant genes of early HSC differentiation and 50 antibodies. To validate differentially expressed surface marker genes, we performed extensive in vitro and in vivo studies of FACS-purified subpopulations of human bone marrow and mobilized peripheral blood samples (mPB) from healthy donors. Our multi-omics based single HSPC profiling indicated Programmed death ligand 2 (PD-L2 / CD273) as a surface marker expressed on the most immature HSCs within the hematopoietic compartment defined as CD34 +CD38 -CD45RA -CD90 +. Prospective FACS-based isolation allowed a deeper understanding of transcriptomic differences depending on PD-L2 positivity. A transcriptomic principal component analysis of CD273 hi and CD273 low expressing HSCs isolated pairwise from four healthy donors proved distinct clustering accompanied by discriminative molecular signatures. Stem cell genes as HOPX, MLLT3 and MPL were enriched in CD273 hi HSCs, and CDK6 was downregulated, further validating our single-cell analysis. The differential expression of stem cell genes was validated at protein level. Single cell tracking using video-microscopy confirmed a delayed entry into cell cycle in CD273 hi HSCs, suggesting deeper quiescence of these cells. Furthermore, their mitochondrial potential was reduced. In vivo transplantations of both populations in immunocompromised NSGs are ongoing and will reveal the long-term repopulation potential of CD273 hi HSCs. Multiple in vitro differentiation assays demonstrated CD273 positivity to be associated with an increased stem cell phenotype with higher expression of EPCR (CD201) and CD90 in liquid culture, a delayed differentiation and high capability of generating multipotent colonies. Interestingly, CD273 surface expression was strongly upregulated in HSCs upon in vitro culture. Since PD-L2 is known as a ligand for the Programmed death receptor 1 (PD1), we investigated the immunomodulating function of PD-L2 expressing HSPCs by establishing an allogeneic lymphocyte reaction assay. In addition to their stem cell-like characteristics, PD-L2 expressing HSPCs mediated diminished T cell proliferation in allogeneic co-culture settings. Furthermore, cytokine profiling of co-culture supernatants indicated increased secretion of anti-inflammatory cytokines (IL-10 and TGF-β1), accompanied by reduced secretion of pro-inflammatory cytokines (IL-17A) and a diminished expression of the pro-inflammatory receptor CD69 on CD8 + T cells. These findings showed that CD34 + cells isolated from mobilized peripheral blood of healthy donors possess immunomodulating capabilities, and that PD-L2 expression indicates an immunocompromising HSC phenotype. In conclusion, this study demonstrates differential surface expression of PD-L2 within the most immature human HSCs. PD-L2 expression is associated with HSC quiescence and delayed in vitro differentiation. Furthermore, these results provide valuable insights into the immunomodulating features of HSCs expressing PD-L2.

Deciphering the Differential Impact of Thrombopoietin/MPL Signaling on Hematopoietic Stem Cell Function in Bone Marrow and Spleen

Introduction Thrombopoietin (TPO) and its receptor MPL are key regulators of hematopoietic stem cell (HSC) function. In humans, ablation of either TPO or MPL leads to bone marrow failure in the early years of life. In contrast, mice lacking TPO or MPL display marked thrombocytopenia and severely reduced HSC numbers but manage to maintain normal survival up to one year of age. In this study, we investigated whether these mice eventually develop marrow failure with longer follow up, with a specific focus on understanding the effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches. Methods Marrow and spleen hematopoiesis in the TPO -/- mice and MPL -/- mice were examined during a 2-yr follow-up. Results At 2 yrs of age, TPO -/- and MPL -/- mice exhibited not only a severely decreased platelet count (TPO -/- vs. MPL -/- vs. control: 38 vs. 107 vs. 1255 x 10 9/L) but also a moderate reduction in neutrophil count compared to age-matched wild-type controls (TPO -/- vs. MPL -/- vs. control: 1.1 vs. 1.7vs.3.3 x 10 3/ul). Femur marrow cell count or spleen cell count was not significantly different between TPO -/- or MPL -/- mice and control mice. There was no significant difference in overall survival between the TPO -/- or MPL -/- mice and wild-type controls. Marrow Lin -cKit +Sca1 +CD150 +CD48 - HSC cell numbers were significantly decreased in young (4-6mo) TPO -/- and MPL -/- mice (~4-fold) compared to age-matched control mice. This HSC loss was further worsened in old (~24mo) TPO -/- (25-fold) and MPL -/- (12-fold) mice compared to control mice. In contrast, there was no significant difference in splenic HSC cell numbers between young TPO -/- or MPL -/- mice and control mice, with only a moderate decrease of HSCs in old TPO -/- and MPL -/- (~4-fold) mice compared to control mice. I n vivo using BrdU labeling revealed an increased proliferation of marrow HSCs in TPO -/- and MPL -/- mice compared to age-matched control mice. In contrast, there was no significant increase in splenic HSC proliferation in TPO -/- or MPL -/- mice. These findings suggest that TPO/MPL signaling has distinct effects on both the number and the function of HSCs in the marrow and spleen. Competitive repopulation assays revealed that HSCs from 2yr old TPO -/- or MPL -/- marrow failed to engraft the recipient mice at 12 weeks post-transplantation. We also conducted competitive repopulation assays in which spleen cells from old TPO -/- mice (CD45.2) were injected together with spleen cells from 2yr old wild-type competitor mice (CD45.1) into lethally irradiated wild-type recipients (CD45.1). At 12 days after transplantation, the TPO -/- spleen donor showed comparable engraftment in the Lin -cKit +Sca1 + stem/progenitor cell compartment, similar to the wild-type competitor spleen donor, suggesting that at least the short-term engraftment capacity of the TPO -/- spleen donor was not impaired. We are currently conducting further investigation to assess the long-term repopulation capacity of TPO -/- spleen HSCs. To investigate how TPO/MPL ablation impacts the hematopoietic niche in the marrow and spleen, we used quantitative flow cytometry and/or fluorescence imaging to examine these niche cells and their expression of niche factors. First, while there was no significant difference in the marrow vascular area (measured by in vivo VE-cadherin labeling) between TPO -/- or MPL -/- mice and control mice, we observed a significant increase in the splenic vascular area in TPO -/- and MPL -/- mice. Second, CXCL12 levels were significantly decreased in marrow endothelial cells (ECs; CD45 -CD31 +) from TPO -/- and MPL -/- mice compared to control mice; in contrast, CXCL12 levels were significantly increased in splenic ECs from TPO -/- and MPL -/- mice. Similar results were also observed with marrow and splenic perivascular stromal cells (CD45 -CD31 -Ter119 -Sca1 -). Therefore, TPO/MPL ablation enhanced both the quantity and quality of the splenic hematopoietic niche, which could contribute to the maintenance of splenic hematopoiesis in TPO -/- and MPL -/- mice. Last, consistent with findings from the TPO/MPL ablated mice, exogenous TPO treatment increased marrow HSC proliferation but did not alter splenic HSC proliferation. In addition, TPO treatment increased CXCL12 expression in marrow ECs, but decreased CXCL12 levels in splenic ECs. Taken together, our study revealed the complexity of TPO/MPL signaling in HSC regulation which differs between the marrow and spleen

Bcor Truncation Mutations Collaborate with a U2AF1 S34F Mutant to Promote Clonal Hematopoiesis and MDS

Mutations in spliceosome genes such as U2AF1 have been identified across major cancer types, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Mutations involving the U2AF1 gene, mostly commonly involving amino acids 34 or 157 have been reported in up to 20% of patients with MDS. To investigate how mutant U2AF1 contribute to hematologic malignancy, we generated transgenic mice that expressed a mutant U2AF1 S34F mutant under control of hematopoietic specific Vav1 regulatory elements. Sanger sequencing demonstrated that expression of the mutant U2AF1 was similar to that of wild-type (WT) U2AF1 in bone marrow and spleen. U2AF1 S34F mice showed decreased long-term hematopoietic stem cells (HSC) compared with age matched wild type WT mice. Hematopoietic stem cell transplantation (HSCT) showed that U2AF1 S34F HSCs repopulated recipients less efficiently than WT HSCs, consistent with previous reports. However, U2AF1 S34F mice did not develop evidence of hematologic malignancy, suggesting that additional mutations were required to develop MDS or AML. BCOR, a corepressor for BCL6, has been reported to be frequently co-mutated in MDS patients with U2AF1 S34F, suggesting that these two mutations might collaborate to produce MDS. To test the hypothesis, we introduced a BCOR truncation mutation into hematopoietic stem and progenitor cells (HSPC) from U2AF1 S34F or WT mice, using CRISPR-Cas9, followed by transplant to WT recipient mice. Primary recipients of U2AF1 S34F and Bcor double mutant bone marrow (U2B) showed decreased engraftment compared to WT-Bcor mutant (WB) recipients, consistent with a competitive disadvantage of U2AF1 S34F HSPC observed previously by several labs. We performed secondary HSCT to evaluate the long-term repopulation pattern of U2B HSPC. Secondary transplant recipients from U2B primary mice showed anemia and leukopenia, consistent with MDS. U2B secondary recipients typically showed low engraftment initially (reflecting ineffective hematopoiesis) followed by gradually increasing proportion of U2B cells in the peripheral blood. At necropsy, all secondary recipients of U2B bone marrow showed evidence of clonal hematopoiesis involving cells with 1 or 2 distinct, unique Bcor truncation mutations. We then sequenced bone marrow DNA from the U2B mouse used as donor for the secondary transplant and identified 14 independent Bcor mutations. All 14 Bcor mutations were out of frame, and encoded a truncated Bcor protein, indicating that cells with a U2AF1 S34F and a Bcor truncation had a competitive advantage in vivo. These results suggest that loss of normal Bcor function can collaborate with a U2AF1 S34F mutant, resulting in clonal expansion of abnormal HSC.

TNFα-Driven Interplay between Hematopoietic Stem Cells and Antigen-Specific Cytotoxic T Cells

Clonal hematopoiesis (CH) is governed by aberrant hematopoietic stem cell (HSC) clones with leukemia-associated somatic mutations that arise spontaneously during organismal aging or after exposure to genotoxic insults. Cells with a somatic mutation can be eliminated through presentation of neoantigens on MHC class I (MHC-I) to antigen-specific cytotoxic T cells. Indeed, many of the CH-related mutations, such as JAK2V617F, are reported or predicted to elicit antigen-specific cytotoxic T cell response. On the other hand, accumulating evidence suggests that inflammatory environment facilitates clonal expansion of abnormal HSCs. However, much remains to be elucidated on whether HSCs expressing such non-self peptides can be eliminated by antigen-specific cytotoxic T cells and how inflammation affects the T cell-mediated HSC regulation. Our RNA-seq analyses revealed that genes involved in MHC-I-dependent antigen presentations, such as Nlrc5, H2-kb and B2m, are most abundantly expressed in HSCs (Lin -/c-Kit +/Sca-1 +/Flk2 -/CD48 -/CD150 +) among murine bone marrow immature hematopoietic cells. Using transgenic mice that ubiquitously express a model antigen ovalbumin (OVA) and a monoclonal antibody that specifically recognizes MHC-I-OVA complex, we found that while MHC-I protein is abundantly expressed on the surface of hematopoietic cells except granulocytes, HSCs and multipotent progenitor cells (MPPs) possess greater capacity to present intracellular antigens through MHC-I compared to myeloid progenitor and mature cells. Remarkably, in vitro co-culture experiments revealed that whereas OVA-expressing (OVA +) HSCs, MPPs and myeloid progenitors could directly activate OVA-specific OT-I CD8 + T cells through MHC-I-dependent antigen presentation, HSCs are the most susceptible population to the killing effect by cytotoxic T cells. Indeed, co-infused OT-I CD8 + T cells totally abolished the long-term repopulation capacity of OVA + HSCs in a serial transplantation setting, and adoptive transfer of OT-I CD8 + T cells to mixed chimeric mice that harbor both wild-type and OVA + hematopoietic cells completely and specifically eliminated hematopoiesis governed by OVA + HSCs. TNFα is not only a prototypical inflammatory cytokine but also one of the major cytokines that are secreted by CD8 + cytotoxic T cells. Notably, genes directly involved in T cell regulation including Cxcl9, Cd274, and Pdcd1lg2 were induced in HSCs, but not myeloid progenitor cells, upon exposure to TNFα (Yamashita and Passegué, Cell Stem Cell 2019). Indeed, upon co-culturing OT-I CD8 + T cells produced TNFα as well as IFNγ and Granzyme B and OVA + HSCs but not myeloid progenitor cells produced CXCL9 largely in a TNFα receptor-dependent manner. Interestingly, such HSC response to T cell-derived TNFα was critical for naïve CD8 + T cells to rapidly differentiate to effector cytotoxic T cells, as TNFα receptor-deficient ( Tnfr1 -/-Tnfr2 -/-) OVA + HSCs stimulated proliferation and cytokine production of OT-I CD8 + T cells but failed to stimulate Granzyme B production, whereas IFNγ receptor deficiency ( Ifngr -/-) did not have such effect. As a result, Tnfr1 -/-Tnfr2 -/- OVA + HSCs, but not Ifngr -/- OVA + HSCs, could escape from the killing by OT-I CD8 +T cells. In sharp contrast, prior exposure to environmental TNFα, but not to IL-1β or IL-6, before interaction with OT-I CD8 + T cells rendered OVA + HSCs resistant to the killing by OT-I CD8 + T cells largely through upregulation of PD-L1 and PD-L2. Of note, upon 24-month aging or acquisition of Jak2V617F mutation, OVA + HSCs still maintained robust MHC-I-dependent antigen presentation activity and high susceptibility to the killing by OT-I CD8 + T cells, though prior exposure to TNFα allowed for their immune escape. Consequently, adoptive transfer of OT-I CD8 + T cells completely and specifically eliminated Jak2V617F-mutant OVA + HSCs and reversed myeloproliferative neoplasms in the mixed chimeric mice. Taken together, our results reveal the robustness of HSC quality control via MHC-I-dependent antigen presentation to antigen-specific cytotoxic T cells. They also highlight a critical but complex role of TNFα in active interplay between HSCs and antigen-specific cytotoxic T cells as well as immune evasion by HSCs during inflammation. Specifically targeting abnormal HSCs by antigen-specific cytotoxic T cells may pave the way to eradicate mutant HSC clones and prevent CH-associated disease.

RNA binding protein SYNCRIP maintains proteostasis and self-renewal of hematopoietic stem and progenitor cells

Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.

Differential expression of endothelial protein C receptor (EPCR) in hematopoietic stem and multipotent progenitor cells in young and old mice

Endothelial protein C receptor (EPCR) has emerged as one of the most conserved and reliable surface markers for the prospective identification and isolation of hematopoietic stem cells (HSCs). Prior studies have consistently demonstrated that EPCR expression enriches HSCs capable of long-term multilineage repopulation in both mouse and human across different hematopoietic tissues, including bone marrow (BM), fetal liver and ex vivo HSC expansion cultures. However, little is known about the expression profiles of EPCR in multipotent progenitor (MPP) populations located immediately downstream of HSCs in the hematopoietic hierarchy and which play a major role in sustaining lifelong blood cell production. Here, we incorporate EPCR antibody detection into a multi-parameter flow cytometric panel, which allows accurate identification of HSCs and five MPP subsets (MPP1-5) in mouse BM. Our data reveal that all MPP populations contain EPCR-expressing cells. Multipotent MPP1 and MPP5 contain higher proportion of EPCR+ cells compared to the more lineage-biased MPP2–4. Notably, high expression of EPCR enriches phenotypic HSC and MPP5, but not MPP1. Comparison of EPCR expression profiles between young and old BM reveals ageing mediated expansion of EPCR-expressing cells only in HSCs, but not in any of the MPP populations. Collectively, our study provides a comprehensive characterization of the surface expression pattern of EPCR in mouse HSC and MPP1–5 cells during normal and aged hematopoiesis.

Timing and regulation of hematopoietic stem cell colonization of the human fetal bone marrow by endothelial and CAR stromal cells during pregnancy

The bone marrow (BM) contains the major reservoir of immature and maturing hematopoietic and immune cells throughout adult life while also harboring most hematopoietic stem and progenitor cells (HSPCs). BM-retained hematopoietic stem cells (HSCs) are mostly maintained in a quiescent, non-motile mode. A small fraction of BM-retained HSPC daily proliferate, differentiate, and migrate to the circulation, to replenish the blood with new immature and maturing blood and (all) immune (both myeloid and lymphoid) cells with a finite life span.1 The anti-coagulation and anti-inflammatory receptor EPCR is also functionally expressed by primitive BM-retained HSCs which are endowed with the highest competitive long-term repopulation potential (LT-HSC). Only BM-retained, quiescent EPCR-positive LT-HSCs are protected from DNA damaging insults including clinical chemotherapy and radiation treatments. Primitive EPCR-positive LT-HSC chemotherapy resistance requires the CXCL12-CXCR4 axis that also regulates HSC quiescence, cell cycle, and directional migration as well as the aPC/EPCR/PAR1 axis.2,3 The chemokine CXCL12 is highly expressed by many BM endothelial and stromal cells types including HSC niche supporting osteoprogenitor cells termed CXCL12 abundant reticular cells (CAR cells), while primitive EPCR-positive fetal liver and adult BM HSC functionally express its major receptor CXCR4.4 Most functional HSC studies involve mice experimental preclinical models as well as results obtained from clinical BM transplantation protocols. During fetal development, HSCs migrate from the fetal liver to the fetal BM and spleen for their lodgment and repopulation. Stem cell homing to the BM during development and in functional experimental transplantation assays including with human HSC in transplanted immune deficient mice is CXCL12/CXCR4-dependent.5,6 The precise timepoint of HSC colonization of the human fetal BM and spleen during pregnancy, as well as the identity of BM stromal niche cells and ligand–receptor interactions which regulate human BM HSC lodgment during fetal development are poorly understood. The group of Zheng et al7 have recently reported on the emergence of HSC in the human fetal BM and spleen and their landscape microenvironment during pregnancy by single-cell RNAseq analysis. This work revealed that functional, human fetal LT-HSC as assayed in transplanted immune deficient mice do not emerge before week 12 post conception. By careful examination of various types of fetal BM stromal and endothelial cells during weeks 10–14 post conception and their transcriptome, this study revealed that fetal BM CAR-like cells endowed with the high levels of CXCL12 production together with fetal endothelial cells which also highly express CXCL12 are the major LT-HSC niche supporting cells. Interestingly, the human fetal spleen was not repopulated with functional human LT-HSC before week 14 post conception, revealing that fetal BM is superior in providing critical signals for LT-HSC homing and lodgment over the fetal spleen. Importantly, self-renewing human CD146 positive osteoprogenitor CAR-like cells were detected in adult human BM and were found to provide a niche for adult human LT-HSC as well as in a miniature human bone implanted in immune deficient mice.8 In conclusion, this study provides evidence for the first functional fetal BM LT-HSC as well as their BM supporting microenvironment and niches.

Ex vivo reprogramming of human hematopoietic stem cells is accompanied by increased transcripts of genes regulating metabolic integrity

The regenerative potential of human hematopoietic stem cells (HSCs) is functionally defined by their ability to provide life-long blood cell production and to repopulate myeloablated allogeneic transplant recipients. The expansion of HSC numbers is dependent not only on HSC divisions but also on a coordinated adaptation of HSCs to metabolic stress. These variables are especially critical during the ex vivo culture of HSCs with cytokine combinations, which frequently results in HSC exhaustion. We have previously reported that human CD34+ hematopoietic stem and progenitor cells (HSPCs) can be efficiently reprogrammed ex vivo and that the number of phenotypic HSCs with long-term repopulation capacity is expanded in the presence of a combination of cytokines and an epigenetic modifier. Here, we present evidence that ex vivo HSC reprogramming and maintenance is accompanied by increased transcripts of genes regulating metabolic integrity, including SIRT1 and SIRT3.

Dlk1 maintains adult mice long-term HSCs by activating Notch signaling to restrict mitochondrial metabolism

BackgroundAdult hematopoietic stem cells (HSCs) homeostasis is critically important in maintaining lifelong hematopoiesis. However, how adult HSCs orchestrate its homeostasis remains not fully understood. Imprinted gene Dlk1 has been shown to play critical role in mouse embryonic hematopoiesis and in regulation of stem cells, but its physiological roles in adult HSCs are unknown.MethodsWe performed gene expression analysis of Dlk1, and constructed conditional Dlk1 knockout (KO) mice by crossing Mx1 cre mice with Dlkflox/flox mice. Western blot and quantitative PCR were used to detect Dlk1 KO efficiency. Flow cytometry was performed to investigate the effects of Dlk1 KO on HSCs, progenitors and linage cells in primary mice. Competitive HSCs transplantation and secondary transplantation was used to examine the effects of Dlk1 KO on long-term hematopoietic repopulation potential of HSCs. RNA-Seq and cell metabolism assays was used to determine the underlying mechanisms.ResultsDlk1 was highly expressed in adult mice long-term HSCs (LT-HSCs) relative to progenitors and mature lineage cells. Dlk1 KO in adult mice HSCs drove HSCs enter active cell cycle, and expanded phenotypical LT-HSCs, but undermined its long-term hematopoietic repopulation potential. Dlk1 KO resulted in an increase in HSCs’ metabolic activity, including glucose uptake, ribosomal translation, mitochondrial metabolism and ROS production, which impaired HSCs function. Further, Dlk1 KO in adult mice HSCs attenuated Notch signaling, and re-activation of Notch signaling under Dlk1 KO decreased the mitochondrial activity and ROS production, and rescued the changes in frequency and absolute number of HSCs. Scavenging ROS by antioxidant N-acetylcysteine could inhibit mitochondrial metabolic activity, and rescue the changes in HSCs caused by Dlk1 KO.ConclusionOur study showed that Dlk1 played an essential role in maintaining HSC homeostasis, which is realized by governing cell cycle and restricting mitochondrial metabolic activity.

Fecal microbiota transplantation from young mice rejuvenates aged hematopoietic stem cells by suppressing inflammation

AbstractHematopoietic stem cell (HSC) aging is accompanied by hematopoietic reconstitution dysfunction, including loss of regenerative and engraftment ability, myeloid differentiation bias, and elevated risks of hematopoietic malignancies. Gut microbiota, a key regulator of host health and immunity, has recently been reported to affect hematopoiesis. However, there is currently limited empirical evidence explaining the direct impact of gut microbiome on aging hematopoiesis. In this study, we performed fecal microbiota transplantation (FMT) from young mice to aged mice and observed a significant increment in lymphoid differentiation and decrease in myeloid differentiation in aged recipient mice. Furthermore, FMT from young mice rejuvenated aged HSCs with enhanced short-term and long-term hematopoietic repopulation capacity. Mechanistically, single-cell RNA sequencing deciphered that FMT from young mice mitigated inflammatory signals, upregulated the FoxO signaling pathway, and promoted lymphoid differentiation of HSCs during aging. Finally, integrated microbiome and metabolome analyses uncovered that FMT reshaped gut microbiota composition and metabolite landscape, and Lachnospiraceae and tryptophan-associated metabolites promoted the recovery of hematopoiesis and rejuvenated aged HSCs. Together, our study highlights the paramount importance of the gut microbiota in HSC aging and provides insights into therapeutic strategies for aging-related hematologic disorders.

BCR::ABL1 -Dependent Promotion of Hematopoietic Stem Cells (HSC) and Progenitors in an Inducible Embryonic Stem Cell (ESC) Model

Our current understanding of the effect of BCR::ABL1 oncogene in leukemia development largely relies on studies using in vitro and in vivo leukemogenesis models. Among them, in vivo models using retrovirus-mediated BCR-ABL gene transfer in murine embryonic stem cell (ESC)-derived hematopoietic cells suggested that the enforced expression of BCR::ABL1 was sufficient to confer them an in vivo long-term repopulation potential. However, in these models, the unique BCR::ABL1 dependence of the generation of this potential has not been shown. Our study aimed at getting a better understanding of the early events regulated by BCR::ABL1 during the course of ESC differentiation without retroviral-mediated gene transfer. The elucidation of these early events could also be of major interest in therapy as generation of bona fide HSCs from normal ESC or iPSC remains a major challenge. To this end, we engineered a unique Tet-ON inducible system in which BCR::ABL1(p210) expression level can be tightly controlled using doxycycline (Dox). As compared to previously reported ESC-derived models, our system contains a single copy of BCR::ABL1 inserted into the murine Col1a1 locus under the control of Tetracycline operator, is sensitive to Dox, leakproof and reversible. The first striking results was the timing of Dox-inducible BCR-ABL expression during the course of ESC differentiation, impacting both the frequency and emergence of hematopoietic progenitors. Indeed, the expression of BCR::ABL1 at day+5 favors significantly the emergence and maintenance of immature hematopoietic progenitors. Importantly, these cells can self-renew only in the presence of Dox. Several hematopoietic stem-like cell lines were established and two of them (HSCL-A and HSCL-C) have been explored in detail. A major BCR-ABL-dependent proliferative ability was documented in both (Figure 1D). Both expressed Lin-Sca-1+c-Kit+CD150+CD48- and eSLAM, corresponding to phenotypically identified adult mouse HSCs. Importantly, these cells exhibited a normal myeloid and erythroid differentiation capacity in CFC assays, giving rise also to CFU-GEMM. We then evaluated the ability of HSCL to self-renew using long-term culture-initiating cell (LTC-IC) assays. To this purpose, EPCR+CD45+/loCD48-CD150+ (ESLAM) cells were sorted at a single cell level and co-cultured on a supportive AFT024 feeder layers for 4 weeks. LTC-IC-derived progenitors were then assessed using CFC assays. The frequency of ESLAM LTC-ICs was 0.53 from adult bone marrow and 0.52 from HSCL lines demonstrating their self-renewal ability. To better characterize HSCL cell lines, we sorted LSK cell population from HSCL#A, HSCL#C, E14.5 fetal liver and murine adult bone marrow and analyzed their transcriptome signatures by RNA-Seq. Unsupervised principal component analysis with the 1333 genes exhibiting the highest variability revealed that HSCL cell lines clustered together and exhibited distinct molecular signature from fetal and adult LSK. This stratification was validated by clustering with the 50 best variable genes. Among them, we found 17 genes highly expressed in HSCL cell lines associated with HSC quiescence (such as NupR1). GSEA analyses confirmed an enrichment of several cell cycle genes as well as genes involved in unfolded protein response (UPR) and autophagy (p <0.001). Thus, we report here for the first time, a unique BCR-ABL inducible ESC-derived hematopoiesis model which demonstrates the ability of BCR-ABL expression to induce a proliferative signal for extended periods of time in highly primitive hematopoietic cells. This novel tool can be used to dissect CML ontogeny both in vitro and in vivo and perform drug screening at the most primitive stem cell level. It can also be of major interest to uncover molecular events and signaling pathways activated during normal ESC-derived hematopoiesis. Such knowledge will open up new avenues to differentiate human iPSC towards bona fide HSCs for potential clinical applications. Figure 1. Establishment of ES cell-derived hematopoietic cell lines expressing BCR::ABLp210.(A) Representation of the timeline of ES cell differentiation protocol and doxycycline induction. (B) HSCL#A cell morphology. (C) MGG staining of HSCL#A cells. (D) Proliferation curves of HSCL#A and HSCL#C cell lines over 40 days in culture. (E)Kinetics of the effect of doxycycline removal on HSCL#A cells cultured on OP9 cells for 4 consecutive days. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Assessing the status of the Green Peafowl’s “expected stronghold” in dry forests along the Salawin River, north-west Thailand

Summary The suitable habitats of the Endangered Green Peafowl Pavo muticus have declined by 80% over the past century due to extensive anthropogenic degradation. Currently, only six strongholds remain in mainland South-east Asia. While there are estimates of the species’ presence and status in five of these, the sixth one, defined as an “expected stronghold” located in the suitable dry forest along the Salawin River between Thailand and Myanmar, is not well documented. This study assessed the status of the area as a stronghold for the Green Peafowl’s long-term survival by estimating 1) the population density, 2) the current extension of suitable habitats, and 3) the threats to its survival. The area had an estimated density of 0.27 calling males/km2 (CI = 0.07–1.01) inhabiting 9,154 km2 of a mosaic of forest types, including mixed dry pine, mixed deciduous, and dry dipterocarp forests. Higher estimates were reported in other strongholds, including 0.8 birds/km2 in Bago Yoma (Myanmar), 15.8 birds/km2 in north-east Thailand, 1.13–11.34 birds/km2 in HuaiKhaKhaeng Wildlife Sanctuary (Thailand), 0.15–1.7 birds/km2 in northern Cambodia, and 0.15–4.69 birds/km2 in eastern Cambodia / south-central Vietnam. Hunting, habitat disturbance, and the presence of humans posed the greatest danger to the species across the surveyed area. Our results confirm the potential of the area for the species’ long-term survival. However, this “expected stronghold,” which could sustain a population of over 10 calling males/km2 like other high-quality strongholds, is in dire need of a comprehensive management plan to help reduce anthropogenic pressure. Ultimately, transboundary management between Thailand and Myanmar is crucial for the long-term repopulation of this stronghold.

MYB orchestrates T cell exhaustion and response to checkpoint inhibition

CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality—which is referred to as T cell exhaustion1,2—is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1− exhausted effector T cells3–6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.

The Adrenergic Receptor Antagonist Carvedilol Elicits Anti-Tumor Responses in Uveal Melanoma 3D Tumor Spheroids and May Serve as Co-Adjuvant Therapy with Radiation.

Simple SummaryDecades of research efforts aiming to identify a new therapy for reducing mortality rates in metastatic uveal melanoma (UM) have not been successful. While ß-blockers are already used as the gold standard in other tumors, e.g., infantile haemangiomas, UM has not received much attention. In the present study, we investigated ß-blockers to demonstrate their anti-tumor potential for the treatment of UM. Of the ß-blockers tested, carvedilol was able to block tumor cell viability and the long-term survival of the cells. Considering that brachytherapy is one of the most efficient local therapies for UM, the concurrent treatment of carvedilol and irradiation was performed, which resulted in additive effects. The anti-tumor properties of ß-blockers described in this study could lead to a new co-adjuvant treatment of UM with the aim to reduce the rate of metastasis and thus mortality.Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite local tumor control, no effective therapy has been found to prevent metastasis, resulting in a high mortality rate. In the present study, we evaluated the anti-tumor potential of non-selective ß-blockers in 3D tumor spheroids grown from UM cell lines. Of the various ß-blockers tested, carvedilol and its enantiomers were most potent in decreasing the viability of Mel270 spheroids. Carvedilol at a concentration of 10–50 µM significantly elicited cytotoxicity and induced apoptosis in spheroid cells. In result, carvedilol inhibited tumor spheroid growth and compactness, and furthermore prevented the long-term survival and repopulation of spreading spheroid cells. The drug sensitivity of the different spheroids grown from Mel270, 92-1, UPMD2, or UPMM3 cell lines was dependent on 3D morphology rather than on high-risk cytogenetic profile or adrenergic receptor expression levels. In fact, the monosomy-3-containing UPMM3 cell line was most responsive to carvedilol treatment compared to the other cell lines. The concurrent treatment of UPMM3 spheroids with carvedilol and 5 or 10 Gy irradiation revealed additive cytotoxic effects that provided tumor control. Collectively, our data demonstrate the anti-tumor properties of carvedilol and its enantiomers, which may serve as candidates for the co-adjuvant therapy of UM.

Hematopoietic stem cell regeneration through paracrine regulation of the Wnt5a/Prox1 signaling axis.

The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited β-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.

Expansion of Quiescent Hematopoietic Stem Cells under Stress and Nonstress Conditions in Mice.

Hematopoietic stem cells (HSCs) are maintained in the quiescent state for protection from stress. How quiescent HSCs expand in vivo under stress and nonstress conditions, however, is poorly understood. Using the fluorescent ubiquitination-based cell cycle indicator (Fucci) mice, we analyzed quiescent and cycling HSCs in the bone marrow after transplantation and during development and aging. The cell cycle of HSCs in Fucci mice were analyzed by flow cytometry. Single-cell colony assays suggested that cycling cells were likely in the process of differentiation. Long-term competitive repopulation and limiting dilution assays revealed that given a higher frequency of functional HSCs in quiescent cells, durable self-renewal potential was greater in quiescent cells than cycling cells. In the bone marrow,functional HSC pool, represented by quiescent HSCs, was rapidly re-established by three weeks after transplantation, significantly expanded by three weeks of age in development, and gradually accumulated with aging. Single-cell RNA-sequencing with flow cytometric index sorting suggested that high levels of CD201 and Sca-1 expression and a low level of mitochondrial activity were associated with quiescent HSCs. A set of candidate quiescent genes in HSCs were also provided. This study implied that controlling quiescence in HSCs is important for their in vivo expansion and maintenance.

Defects in Long-Term APC Repopulation Ability of Adult Human Bone Marrow Hematopoietic Stem Cells (HSCs) Compared with Fetal Liver HSCs.

Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.