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1.
Nat Methods ; 21(5): 868-881, 2024 May.
Article in English | MEDLINE | ID: mdl-38374263

ABSTRACT

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


Subject(s)
Cell Differentiation , Hematopoiesis , Induced Pluripotent Stem Cells , Organoids , Humans , Organoids/cytology , Organoids/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Animals , Mice , Hematopoietic Stem Cells/cytology , Bone Marrow/metabolism , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cell Culture Techniques/methods , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism
2.
Nature ; 594(7862): 265-270, 2021 06.
Article in English | MEDLINE | ID: mdl-34040261

ABSTRACT

Fast and reliable detection of patients with severe and heterogeneous illnesses is a major goal of precision medicine1,2. Patients with leukaemia can be identified using machine learning on the basis of their blood transcriptomes3. However, there is an increasing divide between what is technically possible and what is allowed, because of privacy legislation4,5. Here, to facilitate the integration of any medical data from any data owner worldwide without violating privacy laws, we introduce Swarm Learning-a decentralized machine-learning approach that unites edge computing, blockchain-based peer-to-peer networking and coordination while maintaining confidentiality without the need for a central coordinator, thereby going beyond federated learning. To illustrate the feasibility of using Swarm Learning to develop disease classifiers using distributed data, we chose four use cases of heterogeneous diseases (COVID-19, tuberculosis, leukaemia and lung pathologies). With more than 16,400 blood transcriptomes derived from 127 clinical studies with non-uniform distributions of cases and controls and substantial study biases, as well as more than 95,000 chest X-ray images, we show that Swarm Learning classifiers outperform those developed at individual sites. In addition, Swarm Learning completely fulfils local confidentiality regulations by design. We believe that this approach will notably accelerate the introduction of precision medicine.


Subject(s)
Blockchain , Clinical Decision-Making/methods , Confidentiality , Datasets as Topic , Machine Learning , Precision Medicine/methods , COVID-19/diagnosis , COVID-19/epidemiology , Disease Outbreaks , Female , Humans , Leukemia/diagnosis , Leukemia/pathology , Leukocytes/pathology , Lung Diseases/diagnosis , Machine Learning/trends , Male , Software , Tuberculosis/diagnosis
3.
J Pediatr Hematol Oncol ; 44(1): e62-e67, 2022 01 01.
Article in English | MEDLINE | ID: mdl-33560082

ABSTRACT

Severe congenital neutropenia (SCN) is a rare disease. Autosomal recessive forms of SCN are more frequent in countries where consanguineous marriages are common. In this report, we describe a 54-day-old female with neutropenia who presented with ecthyma gangrenosum. Clinical exome sequencing was used to identify the mutation. HAX1 messenger RNA and isoforms were examined by real-time quantitative and conventional polymerase chain reaction. Bone marrow aspiration was stained by hematoxylin and eosin. Granulocytes were tested for apoptosis upon H2O2 exposure. T-cell proliferation was tested by flow cytometry. Clinical exome sequencing revealed a novel homozygous acceptor splice site mutation in intron 3 of HAX1 (c.505-1G>C), which reduced both isoforms A and B of HAX1 messenger RNA. The Western blot studies showed a complete absence of HAX1 protein. The purified neutrophils from the patient showed increased apoptosis upon H2O2 exposure, whereas T-cell proliferative responses to various stimuli were intact. The patient was treated with combined antibiotics, filgrastim, and placed on antibiotics prophylaxis. To the best of our knowledge, our data provide the first experimental evidence for HAX1 deficiency because of a splice site mutation. Although 3 other splice site variants have been deposited in databases, functional studies were missing. This novel variant of HAX1 may explain the SCN and secondary infections in our patients.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Congenital Bone Marrow Failure Syndromes/genetics , Introns , Mutation , Neutropenia/congenital , RNA Splice Sites , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Congenital Bone Marrow Failure Syndromes/metabolism , Humans , Hydrogen Peroxide/pharmacology , Infant , Male , Neutropenia/genetics , Neutropenia/metabolism , T-Lymphocytes/metabolism
4.
J Allergy Clin Immunol ; 141(3): 1060-1073.e3, 2018 03.
Article in English | MEDLINE | ID: mdl-28629746

ABSTRACT

BACKGROUND: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA-ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N-terminal truncations in IκBα that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA-ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in IκBα have impaired noncanonical NF-κB activity and defective lymphorganogenesis. OBJECTIVE: We sought to establish genotype-phenotype correlation in patients with AD EDA-ID. METHODS: A disease severity scoring system was devised. Stability of IκBα mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF-κB signaling in skin-derived fibroblasts. RESULTS: Disease severity was greater in patients with IκBα point mutations than in those with truncation mutations. IκBα point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF-κB-dependent IL-6 secretion and upregulation of the NF-κB subunit 2/p100 and RELB proto-oncogene, NF-κB subunit (RelB) components of the noncanonical NF-κB pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF-κB-driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus α-lymphotoxin ß receptor-stimulated fibroblasts from patients with point mutations compared with those with truncations. CONCLUSIONS: IκBα point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF-κB activity in patients with AD EDA-ID.


Subject(s)
Ectodermal Dysplasia/genetics , Ectodermal Dysplasia/immunology , Genetic Diseases, X-Linked/genetics , Genetic Diseases, X-Linked/immunology , Genotype , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Signal Transduction , Carrier Proteins/genetics , Carrier Proteins/immunology , Chemokine CCL20/genetics , Chemokine CCL20/immunology , Ectodermal Dysplasia/pathology , Genetic Diseases, X-Linked/pathology , HEK293 Cells , Humans , Immunologic Deficiency Syndromes/pathology , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/immunology , Interleukin-6/genetics , Interleukin-6/immunology , NF-KappaB Inhibitor alpha/genetics , NF-KappaB Inhibitor alpha/immunology , NF-kappa B p52 Subunit/genetics , NF-kappa B p52 Subunit/immunology , Point Mutation , Primary Immunodeficiency Diseases , Proto-Oncogene Mas , Signal Transduction/genetics , Signal Transduction/immunology
5.
J Clin Immunol ; 38(7): 787-793, 2018 10.
Article in English | MEDLINE | ID: mdl-30255293

ABSTRACT

PURPOSE: Inborn errors of IFN-γ-mediated immunity underlie Mendelian Susceptibility to Mycobacterial Disease (MSMD), which is characterized by an increased susceptibility to severe and recurrent infections caused by weakly virulent mycobacteria, such as Bacillus Calmette-Guérin (BCG) vaccines and environmental, nontuberculous mycobacteria (NTM). METHODS: In this study, we investigated four patients from four unrelated consanguineous families from Isfahan, Iran, with disseminated BCG disease. We evaluated the patients' whole blood cell response to IL-12 and IFN-γ, IL-12Rß1 expression on T cell blasts, and sequenced candidate genes. RESULTS: We report four patients from Isfahan, Iran, ranging from 3 months to 26 years old, with impaired IL-12 signaling. All patients suffered from BCG disease. One of them presented mycobacterial osteomyelitis. By Sanger sequencing, we identified three different types of homozygous mutations in IL12RB1. Expression of IL-12Rß1 was completely abolished in the four patients with IL12RB1 mutations. CONCLUSIONS: IL-12Rß1 deficiency was found in the four MSMD Iranian families tested. It is the first report of an Iranian case with S321* mutant IL-12Rß1 protein. Mycobacterial osteomyelitis is another type of location of BCG infection in an IL-12Rß1-deficient patient, notified for the first time in this study.


Subject(s)
Disease Susceptibility , Interleukin-12/metabolism , Interleukin-23/metabolism , Mycobacterium Infections/etiology , Mycobacterium Infections/metabolism , Receptors, Interleukin-12/deficiency , Adolescent , Adult , Alleles , BCG Vaccine/immunology , Biomarkers , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay , Female , Genetic Predisposition to Disease , Genotype , Humans , Immunophenotyping , Infant , Iran/epidemiology , Male , Mutation , Mycobacterium Infections/epidemiology , Mycobacterium Infections/prevention & control , Prognosis , Young Adult
7.
Nat Genet ; 49(5): 742-752, 2017 May.
Article in English | MEDLINE | ID: mdl-28369036

ABSTRACT

We identify SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2), also known as BAF60b (BRG1/Brahma-associated factor 60b), as a critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying patients from three unrelated pedigrees characterized by neutropenia, specific granule deficiency, myelodysplasia with excess of blast cells, and various developmental aberrations, we identified three homozygous loss-of-function mutations in SMARCD2. Using mice and zebrafish as model systems, we showed that SMARCD2 controls early steps in the differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts with the transcription factor CEBPɛ and controls expression of neutrophil proteins stored in specific granules. Defective expression of SMARCD2 leads to transcriptional and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. In summary, SMARCD2 is a key factor controlling myelopoiesis and is a potential tumor suppressor in leukemia.


Subject(s)
Cell Differentiation/genetics , Gene Regulatory Networks , Neutrophils/metabolism , Transcription Factors/genetics , Animals , Animals, Genetically Modified , Base Sequence , Cell Line, Tumor , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone , DNA Mutational Analysis , Family Health , Female , Humans , Leukemia, Promyelocytic, Acute/genetics , Leukemia, Promyelocytic, Acute/pathology , Male , Mice, Inbred C57BL , Mice, Knockout , Pedigree , Zebrafish
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