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1.
Cell ; 155(7): 1507-20, 2013 Dec 19.
Article in English | MEDLINE | ID: mdl-24360274

ABSTRACT

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.


Subject(s)
B-Lymphocytes/metabolism , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Promoter Regions, Genetic , Regulon , Animals , Cell Lineage , Cells, Cultured , CpG Islands , DNA Methylation , Genetic Techniques , Mice , Organ Specificity , RNA, Long Noncoding/genetics , Transcription Factors/metabolism , Transcription, Genetic
2.
Stem Cells ; 41(7): 685-697, 2023 Jul 14.
Article in English | MEDLINE | ID: mdl-37220178

ABSTRACT

Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human-induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation, and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal, and MAPK signaling pathways by stage-specific addition of small-molecule regulators CHIR99021, SB431542, and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable de novo generation of human HSPCs with functionality in vivo.


Subject(s)
Hemangioblasts , Induced Pluripotent Stem Cells , Humans , Induced Pluripotent Stem Cells/metabolism , Activins/metabolism , Cell Differentiation , Signal Transduction
3.
Brain ; 146(1): 278-294, 2023 01 05.
Article in English | MEDLINE | ID: mdl-35867854

ABSTRACT

Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wild-type and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.


Subject(s)
Induced Pluripotent Stem Cells , Muscular Atrophy, Spinal , Humans , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Induced Pluripotent Stem Cells/metabolism , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy , Mitochondria/metabolism , Adenosine Triphosphate/metabolism
4.
Biochem Biophys Res Commun ; 642: 1-10, 2023 01 29.
Article in English | MEDLINE | ID: mdl-36535215

ABSTRACT

BLOC1S1 is a common component of BLOC and BORC multiprotein complexes which play distinct roles in endosome and lysosome biology. Recent human mutations in BLOC1S1 associate with juvenile leukodystrophy. As leukodystrophy is linked to perturbed lysosomal lipid storage we explored whether BLOC1S1 itself modulates this biology. Given the central role of the liver in lipid storage, our investigations were performed in hepatocyte specific liver bloc1s1 knockout (LKO) mice and in human hepatocyte-like lines (HLCs) derived from inducible pluripotential stem cells (iPSCs) from a juvenile leukodystrophy subject's with bloc1s1 mutations and from isogenic corrected iPSCs. Here we show that hepatocyte lipid stores are diminished in parallel with increased lysosomal content, increased lysosomal lipid uptake and lipolysis in LKO mice. The lysosomal lipolysis program was independent of macro- and chaperone-mediated lipophagy but dependent on cellular lysosome content. In parallel, genetic induction of lysosomal biogenesis in a transformed hepatocyte cell line replicated depletion of intracellular lipid stores. Interestingly bloc1s1 mutant and isogenic corrected HLCs both showed normal lysosomal enzyme activity. However, relative to the isogenic corrected HLCs, mutant bloc1s1 HLCs showed reduced lysosomal content and increased lipid storage. Together these data show distinct phenotypes in human mutant HLCs compared to murine knockout cells. At the same time, human blcs1s1 mutation and murine hepatocyte bloc1s1 depletion disrupt lysosome content and the cellular lipid storage. These data support that BLOC1S1 modulates lysosome content and lipid handling independent of autophagy and show that lysosomal lipolysis is dependent on the cellular content of functional lysosomes.


Subject(s)
Lipid Metabolism Disorders , Lipolysis , Animals , Mice , Humans , Liver/metabolism , Lysosomes/metabolism , Transcription Factors/metabolism , Lipid Metabolism Disorders/metabolism , Autophagy , Lipids , Nerve Tissue Proteins/metabolism
5.
Exp Cell Res ; 412(1): 113007, 2022 03 01.
Article in English | MEDLINE | ID: mdl-34990619

ABSTRACT

Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare, lysosomal disorder caused by mutations in a gene encoding iduronate-2-sulfatase (IDS). IDS deficiency results in an accumulation of glycosaminoglycans (GAGs) and secondary accumulations of other lipids in lysosomes. Symptoms of MPS II include a variety of soft and hard tissue problems, developmental delay, and deterioration of multiple organs. Enzyme replacement therapy is an approved treatment for MPS II, but fails to improve neuronal symptoms. Cell-based neuronal models of MPS II disease are needed for compound screening and drug development for the treatment of the neuronal symptoms in MPS II. In this study, three induced pluripotent stem cell (iPSC) lines were generated from three MPS II patient-derived dermal fibroblast cell lines that were differentiated into neural stem cells and neurons. The disease phenotypes were measured using immunofluorescence staining and Nile red dye staining. In addition, the therapeutic effects of recombinant human IDS enzyme, delta-tocopherol (DT), and hydroxypropyl-beta-cyclodextrin (HPBCD) were determined in the MPS II disease cells. Finally, the neural stem cells from two of the MPS II iPSC lines exhibited typical disease features including a deficiency of IDS activity, abnormal glycosaminoglycan storage, and secondary lipid accumulation. Enzyme replacement therapy partially rescued the disease phenotypes in these cells. DT showed a significant effect in reducing the secondary accumulation of lipids in the MPS II neural stem cells. In contrast, HPBCD displayed limited or no effect in these cells. Our data indicate that these MPS II cells can be used as a cell-based disease model to study disease pathogenesis, evaluate drug efficacy, and screen compounds for drug development.


Subject(s)
Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Mucopolysaccharidosis II/drug therapy , Mucopolysaccharidosis II/metabolism , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , 2-Hydroxypropyl-beta-cyclodextrin/therapeutic use , Cell Line , Enzyme Replacement Therapy , Glycosaminoglycans/metabolism , Humans , Iduronate Sulfatase/therapeutic use , Induced Pluripotent Stem Cells/pathology , Lipid Metabolism/drug effects , Models, Neurological , Mucopolysaccharidosis II/pathology , Neural Stem Cells/pathology , Phenotype , Recombinant Proteins/therapeutic use , Tocopherols/therapeutic use
6.
Int J Mol Sci ; 24(5)2023 Feb 27.
Article in English | MEDLINE | ID: mdl-36902031

ABSTRACT

RNA-binding motif 8A (RBM8A) is a core component of the exon junction complex (EJC) that binds pre-mRNAs and regulates their splicing, transport, translation, and nonsense-mediated decay (NMD). Dysfunction in the core proteins has been linked to several detriments in brain development and neuropsychiatric diseases. To understand the functional role of Rbm8a in brain development, we have generated brain-specific Rbm8a knockout mice and used next-generation RNA-sequencing to identify differentially expressed genes (DEGs) in mice with heterozygous, conditional knockout (cKO) of Rbm8a in the brain at postnatal day 17 (P17) and at embryonic day 12. Additionally, we analyzed enriched gene clusters and signaling pathways within the DEGs. At the P17 time point, between the control and cKO mice, about 251 significant DEGs were identified. At E12, only 25 DEGs were identified in the hindbrain samples. Bioinformatics analyses have revealed many signaling pathways related to the central nervous system (CNS). When E12 and P17 results were compared, three DEGs, Spp1, Gpnmb, and Top2a, appeared to peak at different developmental time points in the Rbm8a cKO mice. Enrichment analyses suggested altered activity in pathways affecting cellular proliferation, differentiation, and survival. The results support the hypothesis that loss of Rbm8a causes decreased cellular proliferation, increased apoptosis, and early differentiation of neuronal subtypes, which may lead ultimately to an altered neuronal subtype composition in the brain.


Subject(s)
Brain , Transcriptome , Animals , Mice , Mice, Knockout , Brain/metabolism , RNA-Binding Proteins/metabolism , Signal Transduction
7.
Proc Natl Acad Sci U S A ; 116(39): 19626-19634, 2019 09 24.
Article in English | MEDLINE | ID: mdl-31488712

ABSTRACT

Doxorubicin is a widely used chemotherapeutic agent that causes dose-dependent cardiotoxicity in a subset of treated patients, but the genetic determinants of this susceptibility are poorly understood. Here, we report that a noncanonical tumor suppressor activity of p53 prevents cardiac dysfunction in a mouse model induced by doxorubicin administered in divided low doses as in the clinics. While relatively preserved in wild-type (p53+/+ ) state, mice deficient in p53 (p53-/- ) developed left ventricular (LV) systolic dysfunction after doxorubicin treatment. This functional decline in p53-/- mice was associated with decreases in cardiac oxidative metabolism, mitochondrial mass, and mitochondrial genomic DNA (mtDNA) homeostasis. Notably, mice with homozygous knockin of the p53 R172H (p53172H/H ) mutation, which like p53-/- state lacks the prototypical tumor suppressor activities of p53 such as apoptosis but retains its mitochondrial biogenesis capacity, showed preservation of LV function and mitochondria after doxorubicin treatment. In contrast to p53-null state, wild-type and mutant p53 displayed distinct mechanisms of transactivating mitochondrial transcription factor A (TFAM) and p53-inducible ribonucleotide reductase 2 (p53R2), which are involved in mtDNA transcription and maintenance. Importantly, supplementing mice with a precursor of NAD+ prevented the mtDNA depletion and cardiac dysfunction. These findings suggest that loss of mtDNA contributes to cardiomyopathy pathogenesis induced by doxorubicin administered on a schedule simulating that in the clinics. Given a similar mtDNA protection role of p53 in doxorubicin-treated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, the mitochondrial markers associated with cardiomyopathy development observed in blood and skeletal muscle cells may have prognostic utility.


Subject(s)
Cardiotoxicity/metabolism , Cardiotoxicity/prevention & control , Doxorubicin/toxicity , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis/physiology , Cardiomyopathies/metabolism , DNA, Mitochondrial/genetics , DNA-Binding Proteins , Heart Diseases/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/metabolism , Mitochondrial Proteins , Mutation , Myocytes, Cardiac/metabolism , Organelle Biogenesis , Primary Cell Culture , Transcription Factors , Tumor Suppressor Protein p53/genetics
8.
Nature ; 515(7527): 414-8, 2014 Nov 20.
Article in English | MEDLINE | ID: mdl-25132547

ABSTRACT

Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders, and 'a disease of synapses' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies, little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare, multiply affected, large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and, furthermore, dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders.


Subject(s)
Induced Pluripotent Stem Cells/pathology , Mental Disorders/pathology , Synapses/pathology , Animals , Cell Differentiation , Fibroblasts , Glutamine/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Male , Mental Disorders/genetics , Mental Disorders/metabolism , Mice , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Neurons/pathology , Pedigree , Presynaptic Terminals/metabolism , Presynaptic Terminals/pathology , Prosencephalon/metabolism , Prosencephalon/pathology , Protein Binding , Synapses/metabolism , Transcriptome
10.
Mol Ther ; 25(2): 321-330, 2017 02 01.
Article in English | MEDLINE | ID: mdl-28153086

ABSTRACT

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91phox expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.


Subject(s)
Gene Expression Regulation , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/metabolism , Induced Pluripotent Stem Cells/metabolism , Introns , Membrane Glycoproteins/genetics , NADPH Oxidases/genetics , Targeted Gene Repair , Cell Differentiation/genetics , Cell Line , Exons , Gene Editing , Gene Order , Gene Targeting , Gene Transfer Techniques , Genetic Loci , Genetic Vectors , Granulocytes/cytology , Granulocytes/metabolism , Granulomatous Disease, Chronic/therapy , Humans , Mutation , NADPH Oxidase 2 , Transgenes
11.
Mol Ther ; 25(1): 44-53, 2017 01 04.
Article in English | MEDLINE | ID: mdl-28129126

ABSTRACT

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies.


Subject(s)
Cell Differentiation , Gene Editing , Gene Expression , Germ Layers/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Transgenes , Animals , Biomarkers , CRISPR-Cas Systems , Cellular Reprogramming , Gene Targeting , Genetic Loci , Germ Layers/embryology , Macaca mulatta , Organ Specificity/genetics
12.
Blood ; 126(5): 629-39, 2015 Jul 30.
Article in English | MEDLINE | ID: mdl-26041741

ABSTRACT

Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.


Subject(s)
Immunotherapy, Adoptive/methods , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Receptors, Cytokine/antagonists & inhibitors , T-Lymphocytes/immunology , Animals , Antigens, CD19/metabolism , Cell Line, Tumor , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/therapeutic use , Xenograft Model Antitumor Assays
13.
Stem Cells ; 33(12): 3504-18, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26235913

ABSTRACT

The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile, by RNA-seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures.


Subject(s)
Antigens, Differentiation/biosynthesis , Cell Differentiation , Eye Proteins/biosynthesis , Human Embryonic Stem Cells/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Transcriptome , Cell Line , Human Embryonic Stem Cells/cytology , Humans , Retinal Cone Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/cytology
14.
Mol Ther ; 23(1): 147-57, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25288370

ABSTRACT

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.


Subject(s)
Dependovirus/genetics , Granulomatous Disease, Chronic/therapy , Hematopoietic Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , NADPH Oxidases/genetics , Acetobacteraceae/growth & development , Acetobacteraceae/immunology , Cell Differentiation , Gene Expression , Genetic Therapy/methods , Genetic Vectors , Genotype , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/metabolism , Granulomatous Disease, Chronic/pathology , Hematopoietic Stem Cells/pathology , Humans , Induced Pluripotent Stem Cells/pathology , Macrophages/immunology , Macrophages/microbiology , Macrophages/pathology , NADPH Oxidases/metabolism , Neutrophils/immunology , Neutrophils/microbiology , Neutrophils/pathology , Staphylococcus aureus/growth & development , Staphylococcus aureus/immunology , Zinc Fingers/genetics
15.
Stem Cell Res ; 77: 103429, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38703666

ABSTRACT

Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in JAG1 or less frequently, mutations in NOTCH2. The disease has been difficult to diagnose and treat due to variable expression. The generation of this iPSC line (TRNDi036-A) carrying a heterozygous mutation (p.Cys693*) in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.


Subject(s)
Alagille Syndrome , Heterozygote , Induced Pluripotent Stem Cells , Jagged-1 Protein , Mutation , Alagille Syndrome/genetics , Humans , Jagged-1 Protein/genetics , Jagged-1 Protein/metabolism , Induced Pluripotent Stem Cells/metabolism , Cell Line , Male , Female
16.
Cell Stem Cell ; 31(7): 974-988.e5, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38843830

ABSTRACT

Cellular therapies with cardiomyocytes produced from induced pluripotent stem cells (iPSC-CMs) offer a potential route to cardiac regeneration as a treatment for chronic ischemic heart disease. Here, we report successful long-term engraftment and in vivo maturation of autologous iPSC-CMs in two rhesus macaques with small, subclinical chronic myocardial infarctions, all without immunosuppression. Longitudinal positron emission tomography imaging using the sodium/iodide symporter (NIS) reporter gene revealed stable grafts for over 6 and 12 months, with no teratoma formation. Histological analyses suggested capability of the transplanted iPSC-CMs to mature and integrate with endogenous myocardium, with no sign of immune cell infiltration or rejection. By contrast, allogeneic iPSC-CMs were rejected within 8 weeks of transplantation. This study provides the longest-term safety and maturation data to date in any large animal model, addresses concerns regarding neoantigen immunoreactivity of autologous iPSC therapies, and suggests that autologous iPSC-CMs would similarly engraft and mature in human hearts.


Subject(s)
Induced Pluripotent Stem Cells , Macaca mulatta , Myocytes, Cardiac , Animals , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Cell Differentiation , Humans , Transplantation, Autologous , Positron-Emission Tomography , Time Factors , Myocardial Infarction/therapy , Myocardial Infarction/pathology
17.
Blood ; 118(17): 4599-608, 2011 Oct 27.
Article in English | MEDLINE | ID: mdl-21881051

ABSTRACT

Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated ß-globin alleles (ß(s)/ß(s)). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the ß(s) locus, we achieved precise conversion of 1 mutated ß(s) to the wild-type ß(A) in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected ß(s)/ß(A) heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.


Subject(s)
Anemia, Sickle Cell/genetics , Genetic Therapy/methods , Induced Pluripotent Stem Cells/metabolism , Mutagenesis, Site-Directed , Point Mutation , beta-Globins/genetics , Adult , Anemia, Sickle Cell/pathology , Animals , Base Sequence , Cells, Cultured , Coculture Techniques , Feasibility Studies , Humans , Induced Pluripotent Stem Cells/pathology , Mice , Molecular Sequence Data , Point Mutation/physiology , Sequence Homology, Nucleic Acid , beta-Globins/metabolism
18.
Blood ; 117(21): 5561-72, 2011 May 26.
Article in English | MEDLINE | ID: mdl-21411759

ABSTRACT

We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD), a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production, reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs, with subsequent selection and full characterization to ensure no off-target changes, holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the "safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs.


Subject(s)
Granulomatous Disease, Chronic/therapy , Induced Pluripotent Stem Cells/metabolism , Membrane Glycoproteins/physiology , NADPH Oxidases/deficiency , Neutrophils/enzymology , Adult , Animals , Blotting, Southern , Blotting, Western , Bone Marrow/metabolism , Cell Differentiation , DNA Fingerprinting , Dependovirus/genetics , Flow Cytometry , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/pathology , Humans , Karyotyping , Male , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred NOD , Mice, Nude , NADPH Oxidase 2 , NADPH Oxidases/genetics , NADPH Oxidases/physiology , Phagocytosis , RNA, Messenger/genetics , Reactive Oxygen Species/metabolism , Recombination, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Zinc Fingers
19.
bioRxiv ; 2023 Feb 21.
Article in English | MEDLINE | ID: mdl-36865308

ABSTRACT

Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal and MAPK signaling pathways by stage-specific addition of small molecule regulators CHIR99021, SB431542 and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable de novo generation of human HSPCs with functionality in vivo . Significance Statement: The ability to produce functional HSPCs by differentiation of human iPSCs ex vivo holds enormous potential for cellular therapy of human blood disorders. However, obstacles still thwart translation of this approach to the clinic. In keeping with the prevailing arterial-specification model, we demonstrate that concurrent modulation of WNT, Activin/Nodal and MAPK signaling pathways by stage-specific addition of small molecules during human iPSC differentiation provides a synergy sufficient to promote arterialization of HE and production of HSPCs with features of definitive hematopoiesis. This simple differentiation scheme provides a unique tool for disease modeling, in vitro drug screening and eventual cell therapies.

20.
Stem Cell Res ; 71: 103135, 2023 09.
Article in English | MEDLINE | ID: mdl-37393720

ABSTRACT

Expanded human lymphoblast cells from three different aged healthy individuals, 8-year-old male, 0-year-old newborn (NB) male, and 26-year-old female, were used to generate induced pluripotent stem cell (iPSC) lines TRNDi033-A, TRNDi034-A and TRNDi035-A, respectively, by exogenous expression of five reprogramming factors, human OCT4, SOX2, KLF4, L-MYC and LIN28. The authenticity of established iPSC lines was confirmed by the expressions of stem cell markers, karyotype analysis, embryoid body formation, and scorecard analysis. These iPSC lines could serve as healthy donor controls that are age and sex matched for the studies involving patient-specific iPSCs.


Subject(s)
Induced Pluripotent Stem Cells , Female , Infant, Newborn , Humans , Male , Aged , Child , Induced Pluripotent Stem Cells/metabolism , Cell Differentiation , Kruppel-Like Factor 4 , Lymphocytes , Karyotyping , Cellular Reprogramming
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