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1.
bioRxiv ; 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38979172

ABSTRACT

Adult stem cells play a crucial role in tissue homeostasis and repair through multiple mechanisms. In addition to being able to replace aged or damaged cells, stem cells provide signals that contribute to the maintenance and function of neighboring cells. In the lung, airway basal stem cells also produce cytokines and chemokines in response to inhaled irritants, allergens, and pathogens, which affect specific immune cell populations and shape the nature of the immune response. However, direct cell-to-cell signaling through contact between airway basal stem cells and immune cells has not been demonstrated. Recently, a unique population of intraepithelial airway macrophages (IAMs) has been identified in the murine trachea. Here, we demonstrate that IAMs require Notch signaling from airway basal stem cells for maintenance of their differentiated state and function. Furthermore, we demonstrate that Notch signaling between airway basal stem cells and IAMs is required for antigen-induced allergic inflammation only in the trachea where the basal stem cells are located whereas allergic responses in distal lung tissues are preserved consistent with a local circuit linking stem cells to proximate immune cells. Finally, we demonstrate that IAM-like cells are present in human conducting airways and that these cells display Notch activation, mirroring their murine counterparts. Since diverse lung stem cells have recently been identified and localized to specific anatomic niches along the proximodistal axis of the respiratory tree, we hypothesize that the direct functional coupling of local stem cell-mediated regeneration and immune responses permits a compartmentalized inflammatory response.

2.
Nat Cell Biol ; 26(4): 593-603, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38553595

ABSTRACT

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.


Subject(s)
Intrinsically Disordered Proteins , Intrinsically Disordered Proteins/chemistry , Cyclophilin A/genetics , Cyclophilin A/metabolism , RNA-Binding Proteins , Hematopoietic Stem Cells/metabolism
3.
Nat Genet ; 55(9): 1542-1554, 2023 09.
Article in English | MEDLINE | ID: mdl-37580596

ABSTRACT

Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor-transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis.


Subject(s)
Chromatin , Leukemia , Humans , Chromatin/genetics , Cell Lineage/genetics , Hematopoiesis/genetics , Cell Differentiation/genetics , Transcription Factors/genetics
4.
Cells ; 11(16)2022 08 20.
Article in English | MEDLINE | ID: mdl-36010671

ABSTRACT

The lung epithelium is constantly exposed to harmful agents present in the air that we breathe making it highly susceptible to damage. However, in instances of injury to the lung, it exhibits a remarkable capacity to regenerate injured tissue thanks to the presence of distinct stem and progenitor cell populations along the airway and alveolar epithelium. Mechanisms of repair are affected in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF), a progressive life-threatening disorder characterized by the loss of alveolar structures, wherein excessive deposition of extracellular matrix components cause the distortion of tissue architecture that limits lung function and impairs tissue repair. Here, we review the most recent findings of a study of epithelial cells with progenitor behavior that contribute to tissue repair as well as the mechanisms involved in mouse and human lung regeneration. In addition, we describe therapeutic strategies to promote or induce lung regeneration and the cell-based strategies tested in clinical trials for the treatment of IPF. Finally, we discuss the challenges, concerns and limitations of applying these therapies of cell transplantation in IPF patients. Further research is still required to develop successful strategies focused on cell-based therapies to promote lung regeneration to restore lung architecture and function.


Subject(s)
Idiopathic Pulmonary Fibrosis , Animals , Cell- and Tissue-Based Therapy , Epithelial Cells , Humans , Idiopathic Pulmonary Fibrosis/therapy , Lung , Mice , Stem Cells
5.
Leukemia ; 36(8): 1969-1979, 2022 08.
Article in English | MEDLINE | ID: mdl-35618797

ABSTRACT

Eradicating leukemia requires a deep understanding of the interaction between leukemic cells and their protective microenvironment. The CXCL12/CXCR4 axis has been postulated as a critical pathway dictating leukemia stem cell (LSC) chemoresistance in AML due to its role in controlling cellular egress from the marrow. Nevertheless, the cellular source of CXCL12 in the acute myeloid leukemia (AML) microenvironment and the mechanism by which CXCL12 exerts its protective role in vivo remain unresolved. Here, we show that CXCL12 produced by Prx1+ mesenchymal cells but not by mature osteolineage cells provide the necessary cues for the maintenance of LSCs in the marrow of an MLL::AF9-induced AML model. Prx1+ cells promote survival of LSCs by modulating energy metabolism and the REDOX balance in LSCs. Deletion of Cxcl12 leads to the accumulation of reactive oxygen species and DNA damage in LSCs, impairing their ability to perpetuate leukemia in transplantation experiments, a defect that can be attenuated by antioxidant therapy. Importantly, our data suggest that this phenomenon appears to be conserved in human patients. Hence, we have identified Prx1+ mesenchymal cells as an integral part of the complex niche-AML metabolic intertwining, pointing towards CXCL12/CXCR4 as a target to eradicate parenchymal LSCs in AML.


Subject(s)
Bone Marrow , Leukemia, Myeloid, Acute , Bone Marrow/metabolism , Energy Metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Neoplastic Stem Cells/metabolism , Oxidation-Reduction , Tumor Microenvironment
6.
iScience ; 25(5): 104225, 2022 May 20.
Article in English | MEDLINE | ID: mdl-35494238

ABSTRACT

Understanding the regulation of normal and malignant human hematopoiesis requires comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Here, we develop a tailored bioinformatic pipeline to integrate public and proprietary single-cell RNA sequencing (scRNA-seq) datasets. As a result, we robustly identify for the first time 14 intermediate cell states and 11 stages of differentiation in the endothelial and mesenchymal BM compartments, respectively. Our data provide the most comprehensive description to date of the murine HSC-regulatory microenvironment and suggest a higher level of specialization of the cellular circuits than previously anticipated. Furthermore, this deep characterization allows inferring conserved features in human, suggesting that the layers of microenvironmental regulation of hematopoiesis may also be shared between species. Our resource and methodology is a stepping-stone toward a comprehensive cell atlas of the BM microenvironment.

7.
Cell Stem Cell ; 28(12): 2090-2103.e9, 2021 12 02.
Article in English | MEDLINE | ID: mdl-34551362

ABSTRACT

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.


Subject(s)
Extracellular Vesicles , Hematopoietic Stem Cells , Bone Marrow , Bone Marrow Cells , Hematopoiesis
8.
J Bone Miner Res ; 36(11): 2203-2213, 2021 11.
Article in English | MEDLINE | ID: mdl-34173256

ABSTRACT

The remodeling of the extracellular matrix is a central function in endochondral ossification and bone homeostasis. During secondary fracture healing, vascular invasion and bone growth requires the removal of the cartilage intermediate and the coordinate action of the collagenase matrix metalloproteinase (MMP)-13, produced by hypertrophic chondrocytes, and the gelatinase MMP-9, produced by cells of hematopoietic lineage. Interfering with these MMP activities results in impaired fracture healing characterized by cartilage accumulation and delayed vascularization. MMP-10, Stromelysin 2, a matrix metalloproteinase with high homology to MMP-3 (Stromelysin 1), presents a wide range of putative substrates identified in vitro, but its targets and functions in vivo and especially during fracture healing and bone homeostasis are not well defined. Here, we investigated the role of MMP-10 through bone regeneration in C57BL/6 mice. During secondary fracture healing, MMP-10 is expressed by hematopoietic cells and its maximum expression peak is associated with cartilage resorption at 14 days post fracture (dpf). In accordance with this expression pattern, when Mmp10 is globally silenced, we observed an impaired fracture-healing phenotype at 14 dpf, characterized by delayed cartilage resorption and TRAP-positive cell accumulation. This phenotype can be rescued by a non-competitive transplant of wild-type bone marrow, indicating that MMP-10 functions are required only in cells of hematopoietic linage. In addition, we found that this phenotype is a consequence of reduced gelatinase activity and the lack of proMMP-9 processing in macrophages. Our data provide evidence of the in vivo function of MMP-10 during endochondral ossification and defines the macrophages as the lead cell population in cartilage removal and vascular invasion. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).


Subject(s)
Fracture Healing , Matrix Metalloproteinase 10 , Animals , Cartilage , Chondrocytes , Fracture Healing/genetics , Matrix Metalloproteinase 10/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteogenesis
9.
Am J Respir Cell Mol Biol ; 64(4): 465-476, 2021 04.
Article in English | MEDLINE | ID: mdl-33493092

ABSTRACT

Fibroblast activation includes differentiation to myofibroblasts and is a key feature of organ fibrosis. The Notch pathway has been involved in myofibroblast differentiation in several tissues, including the lung. Here, we identify a subset of collagen-expressing cells in the lung that exhibit Notch3 activity at homeostasis. After injury, this activation increases, being found in αSMA-expressing myofibroblasts in the mouse and human fibrotic lung. Although previous studies suggest a contribution of Notch3 in stromal activation, in vivo evidence of the role of Notch3 in lung fibrosis remains unknown. In this study, we examine the effects of Notch3 deletion in pulmonary fibrosis and demonstrate that Notch3-deficient lungs are protected from lung injury with significantly reduced collagen deposition after bleomycin administration. The induction of profibrotic genes is reduced in bleomycin-treated Notch3-knockout lungs that consistently present fewer αSMA-positive myofibroblasts. As a result, the volume of healthy lung tissue is higher and lung function is improved in the absence of Notch3. Using in vitro cultures of lung primary fibroblasts, we confirmed that Notch3 participates in their survival and differentiation. Thus, Notch3 deficiency mitigates the development of lung fibrosis because of its role in mediating fibroblast activation. Our findings reveal a previously unidentified mechanism underlying lung fibrogenesis and provide a potential novel therapeutic approach to target pulmonary fibrosis.


Subject(s)
Collagen/metabolism , Lung/metabolism , Myofibroblasts/metabolism , Pulmonary Fibrosis/metabolism , Receptor, Notch3/deficiency , Actins/metabolism , Animals , Bleomycin , Cell Differentiation , Cell Survival , Cells, Cultured , Disease Models, Animal , Disease Progression , Humans , Lung/pathology , Lung/physiopathology , Male , Mice, Inbred C57BL , Mice, Knockout , Myofibroblasts/pathology , Phenotype , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/physiopathology , Receptor, Notch3/genetics
11.
Cancers (Basel) ; 12(8)2020 Aug 06.
Article in English | MEDLINE | ID: mdl-32781703

ABSTRACT

Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that hematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour-microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.

12.
Blood ; 136(11): 1303-1316, 2020 09 10.
Article in English | MEDLINE | ID: mdl-32458004

ABSTRACT

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non-caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.


Subject(s)
Aldehyde Oxidoreductases/physiology , Carbolines/pharmacology , Cyclohexylamines/pharmacology , Ferroptosis/drug effects , Hematopoiesis/physiology , Leukemia, Myeloid, Acute/enzymology , Neoplasm Proteins/physiology , Phenylenediamines/pharmacology , Aldehyde Oxidoreductases/genetics , Aldehydes/pharmacology , Animals , Cell Line, Tumor , Cytarabine/administration & dosage , Doxorubicin/administration & dosage , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Lipid Peroxidation , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid-Lymphoid Leukemia Protein/physiology , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Oleic Acid/pharmacology , Oncogene Proteins, Fusion/physiology , Oxidation-Reduction , Oxidative Stress , Phospholipid Hydroperoxide Glutathione Peroxidase/antagonists & inhibitors , Phospholipid Hydroperoxide Glutathione Peroxidase/physiology
13.
Nat Commun ; 10(1): 617, 2019 02 06.
Article in English | MEDLINE | ID: mdl-30728354

ABSTRACT

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired.


Subject(s)
Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/drug effects , Immunoconjugates/pharmacology , Proto-Oncogene Proteins c-kit/immunology , Animals , Bone Marrow/drug effects , Bone Marrow Transplantation , Candida albicans/pathogenicity , Cell Death , Cell Line , Female , Genetic Therapy , Humans , Immunoconjugates/administration & dosage , Mice , Mice, Inbred C57BL , Neoplasms , Tissue Donors
14.
Mol Cell ; 65(5): 832-847.e4, 2017 Mar 02.
Article in English | MEDLINE | ID: mdl-28257700

ABSTRACT

R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.


Subject(s)
DNA/metabolism , Genomic Instability , RNA/metabolism , Replication Protein A/metabolism , Ribonuclease H/metabolism , Transcription, Genetic , Binding Sites , DNA/chemistry , DNA/genetics , HEK293 Cells , HeLa Cells , Humans , Nucleic Acid Conformation , Protein Binding , Protein Interaction Domains and Motifs , RNA/chemistry , RNA/genetics , RNA Interference , Replication Protein A/chemistry , Replication Protein A/genetics , Ribonuclease H/chemistry , Ribonuclease H/genetics , Structure-Activity Relationship , Time Factors , Transfection
16.
Nat Commun ; 8: 14060, 2017 01 09.
Article in English | MEDLINE | ID: mdl-28067246

ABSTRACT

Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations-drug and mutation-compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins.


Subject(s)
Cyclohexylamines/pharmacology , Mutation , RNA Splicing/drug effects , Spiro Compounds/pharmacology , Spliceosomes/drug effects , Splicing Factor U2AF/genetics , Animals , Cell Cycle/drug effects , Cell Proliferation/drug effects , HEK293 Cells , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Transgenic , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Primary Cell Culture , Spliceosomes/genetics , Splicing Factor U2AF/metabolism
17.
Blood ; 129(10): 1260-1269, 2017 03 09.
Article in English | MEDLINE | ID: mdl-27940478

ABSTRACT

Alternative splicing generates a diversity of messenger RNA (mRNA) transcripts from a single mRNA precursor and contributes to the complexity of our proteome. Splicing is perturbed by a variety of mechanisms in cancer. Recurrent mutations in splicing factors have emerged as a hallmark of several hematologic malignancies. Splicing factor mutations tend to occur in the founding clone of myeloid cancers, and these mutations have recently been identified in blood cells from normal, healthy elderly individuals with clonal hematopoiesis who are at increased risk of subsequently developing a hematopoietic malignancy, suggesting that these mutations contribute to disease initiation. Splicing factor mutations change the pattern of splicing in primary patient and mouse hematopoietic cells and alter hematopoietic differentiation and maturation in animal models. Recent developments in this field are reviewed here, with an emphasis on the clinical consequences of splicing factor mutations, mechanistic insights from animal models, and implications for development of novel therapies targeting the precursor mRNA splicing pathway.


Subject(s)
Hematologic Neoplasms/genetics , RNA Splicing Factors/genetics , Animals , Humans , Mutation
18.
Cell ; 167(5): 1310-1322.e17, 2016 11 17.
Article in English | MEDLINE | ID: mdl-27863245

ABSTRACT

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.


Subject(s)
Epigenomics , Hematopoietic Stem Cells/cytology , Animals , Cell Lineage , Clone Cells/cytology , Fluorescence , Hematopoiesis , Inflammation/pathology , Mice , Transcription, Genetic
19.
Nat Biotechnol ; 34(7): 738-45, 2016 07.
Article in English | MEDLINE | ID: mdl-27272386

ABSTRACT

Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45-saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45-SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases.


Subject(s)
DNA Damage/immunology , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/immunology , Leukocyte Common Antigens/immunology , Ribosome Inactivating Proteins, Type 1/genetics , Ribosome Inactivating Proteins, Type 1/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Survival/genetics , Cell Survival/immunology , Cells, Cultured , DNA Damage/genetics , Female , Genetic Enhancement/methods , Immunogenetic Phenomena/genetics , Immunotoxins , Mice , Mice, Inbred C57BL , Saporins
20.
Proc Natl Acad Sci U S A ; 113(7): 1871-6, 2016 Feb 16.
Article in English | MEDLINE | ID: mdl-26831087

ABSTRACT

Tle1 (transducin-like enhancer of split 1) is a corepressor that interacts with a variety of DNA-binding transcription factors and has been implicated in many cellular functions; however, physiological studies are limited. Tle1-deficient (Tle1(Δ/Δ)) mice, although grossly normal at birth, exhibit skin defects, lung hypoplasia, severe runting, poor body condition, and early mortality. Tle1(Δ/Δ) mice display a chronic inflammatory phenotype with increased expression of inflammatory cytokines and chemokines in the skin, lung, and intestine and increased circulatory IL-6 and G-CSF, along with a hematopoietic shift toward granulocyte macrophage progenitor and myeloid cells. Tle1(Δ/Δ) macrophages produce increased inflammatory cytokines in response to Toll-like receptor (TLR) agonists and lipopolysaccharides (LPS), and Tle1(Δ/Δ) mice display an enhanced inflammatory response to ear skin 12-O-tetradecanoylphorbol-13-acetate treatment. Loss of Tle1 not only results in increased phosphorylation and activation of proinflammatory NF-κB but also results in decreased Hes1 (hairy and enhancer of split-1), a negative regulator of inflammation in macrophages. Furthermore, Tle1(Δ/Δ) mice exhibit accelerated growth of B6-F10 melanoma xenografts. Our work provides the first in vivo evidence, to our knowledge, that TLE1 is a major counterregulator of inflammation with potential roles in a variety of inflammatory diseases and in cancer progression.


Subject(s)
Co-Repressor Proteins/physiology , Genes, Tumor Suppressor , Inflammation/physiopathology , NF-kappa B/metabolism , Animals , Co-Repressor Proteins/genetics , Inflammation/metabolism , Mice , Mice, Transgenic
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