ABSTRACT
The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17-CD34+CD43- endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17-CD34+CD43+ blood cells and SOX17+CD34+CD43- endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.
Subject(s)
Blood Cells/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , DNA-Binding Proteins/metabolism , Endothelium/metabolism , Hematopoiesis , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , SOXF Transcription Factors/metabolism , Transcription Factors/metabolism , Yolk Sac/metabolism , Blood Cells/cytology , Cell Differentiation , Cell Lineage , Erythroid Cells/cytology , Erythroid Cells/metabolism , Histone Demethylases/antagonists & inhibitors , Histone Demethylases/metabolism , Humans , Models, Biological , Transcription, GeneticABSTRACT
OBJECTIVES: The aim of this study was to repurpose a drug for the treatment of bipolar depression. METHODS: A gene expression signature representing the overall transcriptomic effects of a cocktail of drugs widely prescribed to treat bipolar disorder was generated using human neuronal-like (NT2-N) cells. A compound library of 960 approved, off-patent drugs were then screened to identify those drugs that affect transcription most similar to the effects of the bipolar depression drug cocktail. For mechanistic studies, peripheral blood mononuclear cells were obtained from a healthy subject and reprogrammed into induced pluripotent stem cells, which were then differentiated into co-cultured neurons and astrocytes. Efficacy studies were conducted in two animal models of depressive-like behaviours (Flinders Sensitive Line rats and social isolation with chronic restraint stress rats). RESULTS: The screen identified trimetazidine as a potential drug for repurposing. Trimetazidine alters metabolic processes to increase ATP production, which is thought to be deficient in bipolar depression. We showed that trimetazidine increased mitochondrial respiration in cultured human neuronal-like cells. Transcriptomic analysis in induced pluripotent stem cell-derived neuron/astrocyte co-cultures suggested additional mechanisms of action via the focal adhesion and MAPK signalling pathways. In two different rodent models of depressive-like behaviours, trimetazidine exhibited antidepressant-like activity with reduced anhedonia and reduced immobility in the forced swim test. CONCLUSION: Collectively our data support the repurposing of trimetazidine for the treatment of bipolar depression.
Subject(s)
Bipolar Disorder , Trimetazidine , Rats , Humans , Animals , Trimetazidine/pharmacology , Trimetazidine/therapeutic use , Bipolar Disorder/drug therapy , Bipolar Disorder/genetics , Transcriptome , Drug Repositioning , Leukocytes, Mononuclear , Disease Models, AnimalABSTRACT
The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We generated heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of SOX9 in critical developmental processes.
Subject(s)
Heterozygote , Homozygote , Induced Pluripotent Stem Cells , SOX9 Transcription Factor , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , SOX9 Transcription Factor/metabolism , SOX9 Transcription Factor/genetics , Cell Line , CRISPR-Cas Systems , Gene Knockout Techniques , Gene Editing , Cell DifferentiationABSTRACT
The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of WT1 in critical developmental processes.
Subject(s)
Homozygote , Induced Pluripotent Stem Cells , WT1 Proteins , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , WT1 Proteins/genetics , WT1 Proteins/metabolism , Cell Line , CRISPR-Cas Systems , Cell Differentiation , Gene Knockout Techniques , Gene EditingABSTRACT
We used gene editing to introduce DNA sequences encoding the tdTomato fluorescent protein into the α -skeletal actin 1 (ACTA1) locus to develop an ACTA1-tdTomato induced pluripotent stem cell reporter line for monitoring differentiation of skeletal muscle. This cell line will be used to better understand skeletal muscle maturation and development in vitro as well as provide a useful tool for drug screening and the evaluation of novel therapeutics for the treatment of skeletal muscle disease.
Subject(s)
CRISPR-Cas Systems , Induced Pluripotent Stem Cells , Red Fluorescent Protein , Humans , CRISPR-Cas Systems/genetics , Induced Pluripotent Stem Cells/metabolism , Actins/genetics , Actins/metabolism , Muscle, Skeletal/metabolismABSTRACT
The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.
Subject(s)
Induced Pluripotent Stem Cells , Humans , Induced Pluripotent Stem Cells/metabolism , Heart , Heterozygote , Homozygote , Phenotype , CRISPR-Cas Systems/genetics , COUP Transcription Factor II/genetics , COUP Transcription Factor II/metabolismABSTRACT
We describe the generation and characterisation of five human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, and pluripotency was validated by flow cytometry and immunofluorescence of pluripotency markers, and their differentiation into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.
Subject(s)
Induced Pluripotent Stem Cells , Adult , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , Kruppel-Like Factor 4 , Cell Differentiation , Cell Line , Cellular ReprogrammingABSTRACT
Uncovering the molecular mechanisms of autism spectrum disorder (autism) necessitates development of relevant experimental models that are capable of recapitulating features of the clinical phenotype. Using non-integrative episomal vectors, peripheral blood mononuclear cells derived from three unrelated individuals diagnosed with autism were reprogrammed to induced pluripotent stem cells (iPSCs). The resultant lines exhibited the expected cellular morphology, karyotype, and evidence of pluripotency. These iPSCs constitute a valuable resource to support investigations of the underlying aetiology of autism.
Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Induced Pluripotent Stem Cells , Humans , Induced Pluripotent Stem Cells/metabolism , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/metabolism , Leukocytes, Mononuclear/metabolism , Karyotype , Cell Differentiation , Cellular ReprogrammingABSTRACT
Cytotoxic antineoplastic therapy is used to treat malignant disease but results in long-term immunosuppression in postpubertal and adult individuals, leading to increased incidence and severity of opportunistic infections. We have previously shown that sex steroid ablation (SSA) reverses immunodeficiencies associated with age and hematopoietic stem cell transplantation in both autologous and allogeneic settings. In this study, we have assessed the effects of SSA by surgical castration on T cell recovery of young male mice following cyclophosphamide treatment as a model for the impact of chemotherapy. SSA increased thymic cellularity, involving all of the thymocyte subsets and early T lineage progenitors. It also induced early repair of damage to the thymic stromal microenvironment, which is crucial to the recovery of a fully functional T cell-based immune system. These functional changes in thymic stromal subsets included enhanced production of growth factors and chemokines important for thymopoiesis, which preceded increases in both thymocyte and stromal cellularity. These effects collectively translated to an increase in peripheral and splenic naive T cells. In conclusion, SSA enhances T cell recovery following cyclophosphamide treatment of mice, at the level of the thymocytes and their stromal niches. This provides a new approach to immune reconstitution following antineoplastic therapy.
Subject(s)
Antineoplastic Agents/toxicity , Cyclophosphamide/toxicity , Gonadal Steroid Hormones/immunology , Orchiectomy , T-Lymphocytes/immunology , Animals , Cell Separation , Flow Cytometry , Male , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , Steroids , T-Lymphocytes/drug effects , Thymus Gland/cytology , Thymus Gland/drug effects , Thymus Gland/immunologyABSTRACT
Clozapine has superior efficacy in the treatment of refractory schizophrenia; however, use of clozapine is limited due to severe side effects, including myocarditis. Using non-integrative Sendai virus, we generated induced pluripotent stem cell lines from peripheral blood mononuclear cells of two patients with refractory schizophrenia, one clozapine-tolerant and one clozapine-induced myocarditis. Both cell lines exhibited a normal karyotype and pluripotency was validated by flow cytometry, immunofluorescence and their ability to differentiate into the three germ layers. These lines can be used to generate 2D and 3D patient-specific human cellular models to identify the mechanism by which clozapine induces myocardial inflammation.
Subject(s)
Antipsychotic Agents , Clozapine , Induced Pluripotent Stem Cells , Myocarditis , Schizophrenia , Antipsychotic Agents/adverse effects , Cell Differentiation , Clozapine/adverse effects , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , Myocarditis/chemically induced , Schizophrenia/drug therapy , Schizophrenia/metabolism , Schizophrenia, Treatment-ResistantABSTRACT
Cytotoxic antineoplastic therapy is widely used in the clinic as a treatment for malignant diseases. The treatment itself, however, leads to long-term depletion of the adaptive immune system, which is more pronounced in older patients, predominantly due to thymic atrophy. We and others have previously shown that withdrawal of sex steroids is able to regenerate the aged thymus and enhance recovery from autologous and allogeneic hematopoietic stem cell transplant. In this study we have examined the effects of sex steroid ablation (SSA) on the recovery of lymphopoiesis in the bone marrow (BM) and thymus following treatment with the chemotherapeutic agent cyclophosphamide (Cy) in middle-aged and old mice. Furthermore, we have also examined the impact of this regeneration on peripheral immunity. SSA enhanced the recovery of BM resident hematopoietic stem cells and lymphoid progenitors and promoted lymphopoiesis. Interestingly, Cy alone caused a profound increase in the recently described common lymphoid progenitor 2 (CLP-2) population in the BM. In the thymus, SSA caused a profound increase in cellularity as well as all intrathymic T-lineage progenitors including early T-lineage progenitors (ETPs) and non-canonical T cell progenitors such as the CLP-2. We also found that these transferred into numerical increases in the periphery with enhanced B and T cell numbers. Furthermore, these lymphocytes were found to have an enhanced functional capacity with no perturbation of the TCR repertoire. Taken together, these results provide the basis for the use of SSA in the clinic to enhance treatment outcomes from cytotoxic antineoplastic therapy.
Subject(s)
Aging/physiology , Antineoplastic Agents/adverse effects , Cyclophosphamide/adverse effects , Gonadal Steroid Hormones/deficiency , Lymphopoiesis/physiology , Animals , Bone Marrow/drug effects , Bone Marrow/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Castration , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Lymphopoiesis/drug effects , Male , Mice , Mice, Inbred C57BL , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , Thymus Gland/cytology , Thymus Gland/drug effects , Thymus Gland/immunologyABSTRACT
We describe the generation and characterization of 5 human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.
Subject(s)
Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/pathology , Leukocytes, Mononuclear/pathology , Adult , Cell Line , Female , Humans , Kruppel-Like Factor 4 , Male , Middle Aged , Young AdultABSTRACT
We have generated and characterized seven human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) from a single family, including unaffected and affected individuals clinically diagnosed with Autism Spectrum Disorder (ASD). The reprogramming of the PBMCs was performed using non-integrative Sendai virus containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. All iPSC lines exhibited a normal karyotype and pluripotency was validated by immunofluorescence, flow cytometry and their ability to differentiate into the three embryonic germ layers. These iPSC lines are a valuable resource to study the molecular mechanisms underlying ASD.
Subject(s)
Autism Spectrum Disorder/metabolism , Induced Pluripotent Stem Cells/cytology , Leukocytes, Mononuclear/cytology , Adult , Cellular Reprogramming/genetics , Cellular Reprogramming/physiology , Female , Flow Cytometry , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Leukocytes, Mononuclear/metabolism , Male , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Sendai virus/genetics , Young AdultABSTRACT
A major underlying cause for aging of the immune system is the structural and functional atrophy of the thymus, and associated decline in T cell genesis. This loss of naïve T cells reduces adaptive immunity to new stimuli and precipitates a peripheral bias to memory cells against prior antigens. Whilst multiple mechanisms may contribute to this process, the temporal alliance of thymic decline with puberty has implicated a causative role for sex steroids. Accordingly ablation of sex steroids induces profound thymic rejuvenation. Although the thymus retains some, albeit highly limited, function in healthy adults, this is insufficient for resurrecting the T cell pool following cytoablative treatments such as chemo- and radiation-therapy and AIDS. Increased risk of opportunistic infections and cancer relapse or appearance, are a direct consequence. Temporary sex steroid ablation may thus provide a clinically effective means to regenerate the thymus and immune system in immunodeficiency states.
Subject(s)
Gonadal Steroid Hormones/immunology , Thymus Gland/immunology , Thymus Gland/pathology , Aging/immunology , Animals , Atrophy , Disease Susceptibility , Humans , Thymus Gland/cytologyABSTRACT
The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.
Subject(s)
Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Homeodomain Proteins/metabolism , Mesonephros/cytology , Mesonephros/embryology , Neovascularization, Physiologic/physiology , Aorta/cytology , Aorta/embryology , Aorta/growth & development , Cell Differentiation/physiology , Cells, Cultured , Embryonic Stem Cells/physiology , Gonads/cytology , Gonads/embryology , Gonads/growth & development , Hematopoietic Stem Cells/physiology , Humans , Mesonephros/growth & developmentABSTRACT
Recent evidence suggests that the decline in resistance to viral infections with age occurs predominantly as a result of a gradual loss of naïve antigen-specific T cells. As such, restoration of the naïve T cell repertoire to levels seen in young healthy adults may improve defence against infection in the aged. We have previously shown that sex steroid ablation (SSA) rejuvenates the ageing thymus and increases thymic export of naïve T cells, but it remains unclear whether T cell responses are improved. Using mouse models of clinically relevant diseases, we now demonstrate that SSA increases the number of naïve T cells able to respond to antigen, thereby enhancing effector responses in aged mice. Specifically, aged mice exhibit a delay in clearing influenza A virus, which correlates with diminished specific cytotoxic activity. This is due to a decreased magnitude of response and not an intrinsic defect in effector T cell function. Upon SSA, aged mice exhibit increased T cell responsiveness that restores efficient viral clearance. We further demonstrate that SSA decreases the incidence of an inducible tumour in aged mice and can potentially increase their responsiveness to a low-dose human papillomavirus vaccine in clearing pre-formed tumours. As thymectomy abrogates the increase in T cell numbers and responsiveness following SSA, we propose that the T cell effects of SSA are dependent on thymic reactivation and subsequent replenishment of the peripheral T cell pool with newly emigrated naïve T cells. These findings have important implications for strategies to improve protection from infection and responsiveness to vaccination in the aged.