ABSTRACT
Emerging contaminants are chemical products that are found in low concentrations, are not regulated by environmental norms, and cause health effects. Among this group of contaminants are parabens, a family of p-hydroxybenzoic acid esters used as preservatives in cosmetics, pharmaceuticals, and food products. Recent research describes parabens as endocrine disruptors that can cause health alterations. Some of the best alternatives for pollutant removal include the adsorption process, which can use materials that are inexpensive, abundant, and susceptible to modifications. In this sense, cellulose can be an option for obtaining materials that can be used in the removal of contaminants. This research investigates the synthesis of benzoic cellulose (MCB) and magnetic cellulose (MCM) as well as its use as an adsorbent for the removal of methylparaben (MP) and butylparaben (BP) from water. Likewise, physicochemical characterization, including Fourier transform infrared (FTIR), scanning electronic microscopy (SEM), energy-dispersive spectroscopy (EDS), X-ray diffraction (XRD), and thermogravimetric analysis (TGA), for both cellulose materials was carried out. Moreover, pseudo-first-order, pseudo-second-order, Elovich, Weber, Morris, and Boyd models were used to investigate the adsorption kinetics. As a result, the pseudo-second-order model was favorable for both modified cellulose and the two parabens assayed. Finally, Freundlich, Langmuir, and Sips adsorption isotherm models were investigated; the Langmuir model was the best for the adsorption isotherm data. The adsorption of methylparaben and butylparaben was in the following order: MCM > MCB. The maximum adsorption capacity of MP and BP for MCM was 9.58 and 12.03 mg g-1, respectively. For instance, the results showed that the modified cellulose adsorbed the parabens physically, which could involve electrostatic attraction, hydrogen bonding, π-π bonding, and hydrophobic interactions.
ABSTRACT
BACKGROUND AIMS: The increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice-compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements. METHODS: MSC,WJ were successfully expanded in 3D and final product characterization was in conformity with Critical Quality Attributes and product specifications previously established for 2D expansion conditions. RESULTS: After 6 days of culture, cell yields in the final product from the 3D cultures (mean 9.48 × 108 ± 1.07 × 107 cells) were slightly lower but comparable with those obtained from 2D surfaces (mean 9.73 × 108 ± 2.36 × 108 cells) after 8 days. In all analyzed batches, viability was >90%. Immunophenotype of MSC,WJ was highly positive for CD90 and CD73 markers and lacked of expression of CD31, CD45 and HLA-DR. Compared with 2D expansions, CD105 was detected at lower levels in 3D cultures due to the harvesting procedure from microcarriers involving trypsin at high concentration, and this had no impact on multipotency. Cells presented normal karyotype and strong immunomodulatory potential in vitro. Sterility, Mycoplasma, endotoxin and adventitious virus were negative in both batches produced. CONCLUSIONS: In summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies.
ABSTRACT
Germline SAMD9 mutations are one of the most common alterations that predispose to pediatric myelodysplastic syndrome (MDS), a clonal disorder characterized by ineffective hematopoiesis, increasing the risk of developing acute myeloid leukemia (AML). Up to date, a disease model to study the role of SAMD9 mutation in MDS is still lacking. Here, we have generated a human induced pluripotent stem cell (hiPSC) line carrying SAMD9mut (p.I1567M), taking advantage of CRISPR/Cas9 system. As a result, the genetic engineered hiPSC line represent a new in vitro disease model to understand the impact of SAMD9 mutation at molecular and cellular level during hematopoiesis.
Subject(s)
Induced Pluripotent Stem Cells , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Humans , Child , Induced Pluripotent Stem Cells/metabolism , CRISPR-Cas Systems/genetics , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Heterozygote , Mutation/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Intracellular Signaling Peptides and Proteins/geneticsABSTRACT
Pediatric acute myeloid leukemia (AML) is a rare and heterogeneous disease that remains the major cause of mortality in children with leukemia. To improve the outcome of pediatric AML we need to gain knowledge on the biological bases of this disease. NUP98-KDM5A (NK5A) fusion protein is present in a particular subgroup of young pediatric patients with poor outcome. We report the generation and characterization of human Embryonic Stem Cell (hESC) clonal lines with inducible expression of NK5A. Temporal control of NK5A expression during hematopoietic differentiation from hESC will be critical for elucidating its participation during the leukemogenic process.
ABSTRACT
Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Patients with AML harboring a constitutively active internal tandem duplication mutation (ITDMUT) in the FMS-like kinase tyrosine kinase (FLT3) receptor generally have a poor prognosis. Several tyrosine kinase/FLT3 inhibitors have been developed and tested clinically, but very few (midostaurin and gilteritinib) have thus far been FDA/EMA-approved for patients with newly diagnosed or relapse/refractory FLT3-ITDMUT AML. Disappointingly, clinical responses are commonly partial or not durable, highlighting the need for new molecules targeting FLT3-ITDMUT AML. Here, we tested EC-70124, a hybrid indolocarbazole analog from the same chemical space as midostaurin with a potent and selective inhibitory effect on FLT3. In vitro, EC-70124 exerted a robust and specific antileukemia activity against FLT3-ITDMUT AML primary cells and cell lines with respect to cytotoxicity, CFU capacity, apoptosis and cell cycle while sparing healthy hematopoietic (stem/progenitor) cells. We also analyzed its efficacy in vivo as monotherapy using two different xenograft models: an aggressive and systemic model based on MOLM-13 cells and a patient-derived xenograft model. Orally disposable EC-70124 exerted a potent inhibitory effect on the growth of FLT3-ITDMUT AML cells, delaying disease progression and debulking the leukemia. Collectively, our findings show that EC-70124 is a promising and safe agent for the treatment of AML with FLT3-ITDMUT.
ABSTRACT
Germline pathogenic variants in BRCA1 confer a high risk of developing breast and ovarian cancer. The BRCA1 exon 11 (formally exon 10) is one of the largest exons and codes for the nuclear localization signals of the corresponding gene product. This exon can be partially or entirely skipped during pre-mRNA splicing, leading to three major in-frame isoforms that are detectable in most cell types and tissue, and in normal and cancer settings. However, it is unclear whether the splicing imbalance of this exon is associated with cancer risk. Here we identify a common genetic variant in intron 10, rs5820483 (NC_000017.11:g.43095106_43095108dup), which is associated with exon 11 isoform expression and alternative splicing, and with the risk of breast cancer, but not ovarian cancer, in BRCA1 pathogenic variant carriers. The identification of this genetic effect was confirmed by analogous observations in mouse cells and tissue in which a loxP sequence was inserted in the syntenic intronic region. The prediction that the rs5820483 minor allele variant would create a binding site for the splicing silencer hnRNP A1 was confirmed by pull-down assays. Our data suggest that perturbation of BRCA1 exon 11 splicing modifies the breast cancer risk conferred by pathogenic variants of this gene.
Subject(s)
Breast Neoplasms/genetics , Exons , Genes, BRCA1 , Genetic Carrier Screening , Genetic Predisposition to Disease , RNA Splicing , Female , Humans , IntronsABSTRACT
Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.
Subject(s)
GATA2 Deficiency , Induced Pluripotent Stem Cells , Myelodysplastic Syndromes , CRISPR-Cas Systems/genetics , GATA2 Transcription Factor/genetics , Heterozygote , Humans , Myelodysplastic Syndromes/geneticsABSTRACT
Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.
Subject(s)
Benzimidazoles/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Epithelial Cells/drug effects , Lung Neoplasms/drug therapy , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Pyrazines/pharmacology , A549 Cells , Animals , Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Molecular Targeted Therapy , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA-Seq , Single-Cell Analysis , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative. METHODS: We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs. RESULTS: Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs. CONCLUSIONS: This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML.
Subject(s)
CD28 Antigens/metabolism , Cell Engineering/methods , Hematopoiesis/genetics , Immunotherapy, Adoptive/methods , Interleukin-3 Receptor alpha Subunit/metabolism , Lymphocytes/metabolism , T-Lymphocytes/metabolism , Animals , Female , Humans , Male , MiceABSTRACT
Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.
ABSTRACT
In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.
Subject(s)
GATA2 Transcription Factor/metabolism , Hematopoiesis , Mesoderm/metabolism , Cell Differentiation , GATA2 Transcription Factor/genetics , Gene Expression Regulation , Hemangioblasts/cytology , Hemangioblasts/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mesoderm/cytology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Protein Binding , Single-Cell AnalysisABSTRACT
We have generated two human induced pluripotent stem cell (iPSC) lines from CD133+ cells isolated from umbilical cord blood (CB) of a female child using non-integrative Sendai virus. Here we describe the complete characterization of these iPSC lines: PRYDi-CB5 and PRYDi-CB40.
Subject(s)
AC133 Antigen/genetics , Cell Line , Induced Pluripotent Stem Cells , Animals , Cell Differentiation , Cellular Reprogramming Techniques , Clone Cells , Fetal Blood/cytology , Genetic Markers , Humans , Karyotype , Mice, Inbred NOD , Mice, SCID , Sendai virusABSTRACT
Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc scid Il2rd tm1Wjl /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.
ABSTRACT
Ribosomal protein (RP) expression in higher eukaryotes is regulated translationally through the 5'TOP sequence. This mechanism evolved to more rapidly produce RPs on demand in different tissues. Here we show that 40S ribosomes, in a complex with the mRNA binding protein LARP1, selectively stabilize 5'TOP mRNAs, with disruption of this complex leading to induction of the impaired ribosome biogenesis checkpoint (IRBC) and p53 stabilization. The importance of this mechanism is underscored in 5q− syndrome, a macrocytic anemia caused by a large monoallelic deletion, which we found to also encompass the LARP1 gene. Critically, depletion of LARP1 alone in human adult CD34+ bone marrow precursor cells leads to a reduction in 5'TOP mRNAs and the induction of p53. These studies identify a 40S ribosome function independent of those in translation that, with LARP1, mediates the autogenous control of 5'TOP mRNA stability, whose disruption is implicated in the pathophysiology of 5q− syndrome.
Subject(s)
Autoantigens/metabolism , Protein Biosynthesis , RNA 5' Terminal Oligopyrimidine Sequence , RNA Stability , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Anemia, Macrocytic/genetics , Anemia, Macrocytic/metabolism , Autoantigens/genetics , Bone Marrow Cells/metabolism , Chromosome Deletion , Chromosomes, Human, Pair 5/genetics , Chromosomes, Human, Pair 5/metabolism , HCT116 Cells , Humans , Multiprotein Complexes , Protein Binding , RNA Interference , RNA, Messenger/genetics , Ribonucleoproteins/genetics , Ribosomal Proteins/genetics , Ribosomes/genetics , Time Factors , Transfection , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , SS-B AntigenABSTRACT
We report the generation-characterization of a fetal liver (FL) B-cell progenitor (BCP)-derived human induced pluripotent stem cell (hiPSC) line CRISPR/Cas9-edited to carry/express a single copy of doxycycline-inducible Cas9 gene in the "safe locus" AAVS1 (iCas9-FL-BCP-hiPSC). Gene-edited iPSCs remained pluripotent after CRISPR/Cas9 genome-edition. Correct genomic integration of a unique copy of Cas9 was confirmed by PCR and Southern blot. Cas9 was robustly and specifically expressed on doxycycline exposure. T7-endonuclease assay demonstrated that iCas9 induces robust gene-edition when gRNAs against hematopoietic transcription factors were tested. This iCas9-FL-BCP-hiPSC will facilitate gene-editing approaches for studies on developmental biology, drug screening and disease modeling.
Subject(s)
B-Lymphocytes/metabolism , Bacterial Proteins/biosynthesis , Dependovirus , Endonucleases/biosynthesis , Gene Expression , Genetic Loci , Induced Pluripotent Stem Cells/metabolism , B-Lymphocytes/cytology , Bacterial Proteins/genetics , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Endonucleases/genetics , Gene Editing , Humans , Induced Pluripotent Stem Cells/cytologyABSTRACT
Bone marrow mesenchymal stem/stromal cells (BM-MSCs) are key components of the hematopoietic niche thought to have a direct role in leukemia pathogenesis. BM-MSCs from patients with acute myeloid leukemia (AML) have been poorly characterized due to disease heterogeneity. We report a functional, genetic, and immunological characterization of BM-MSC cultures from 46 AML patients, stratified by molecular/cytogenetics into low-risk (LR), intermediate-risk (IR), and high-risk (HR) subgroups. Stable MSC cultures were successfully established and characterized from 40 of 46 AML patients irrespective of the risk subgroup. AML-derived BM-MSCs never harbored tumor-specific cytogenetic/molecular alterations present in blasts, but displayed higher clonogenic potential than healthy donor (HD)-derived BM-MSCs. Although HD- and AML-derived BM-MSCs equally provided chemoprotection to AML cells in vitro, AML-derived BM-MSCs were more immunosuppressive/anti-inflammatory, enhanced suppression of lymphocyte proliferation, and diminished secretion of pro-inflammatory cytokines. Multivariate analysis revealed that the level of interleukin-10 produced by AML-derived BM-MSCs as an independent prognostic factor negatively affected overall survival. Collectively our data show that AML-derived BM-MSCs are not tumor related, but display functional differences contributing to therapy resistance and disease evolution.
Subject(s)
Leukemia, Myeloid, Acute/therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Adult , Aged , Bone Marrow Cells/cytology , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Differentiation , Cell Proliferation/drug effects , Cells, Cultured , Cytogenetic Analysis , Cytokines/metabolism , Female , Humans , Immunosuppression Therapy , In Situ Hybridization, Fluorescence , Interleukin-10/metabolism , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Male , Mesenchymal Stem Cells/metabolism , Middle Aged , Phytohemagglutinins/pharmacology , Prognosis , Treatment Outcome , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Coenzyme Q10 (CoQ10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ10 ], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703.
Subject(s)
Ataxia/genetics , Intellectual Disability/genetics , Mitochondria/genetics , Mitochondrial Diseases/genetics , Mitochondrial Proteins/genetics , Muscle Weakness/genetics , Rhabdomyolysis/genetics , Ubiquinone/analogs & derivatives , Ubiquinone/deficiency , Ataxia/enzymology , Ataxia/pathology , CRISPR-Cas Systems , Cell Differentiation , Child, Preschool , Dopaminergic Neurons/cytology , Dopaminergic Neurons/metabolism , Electron Transport Chain Complex Proteins/genetics , Electron Transport Chain Complex Proteins/metabolism , Fatal Outcome , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Editing/methods , Gene Expression , Genes, Lethal , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Intellectual Disability/enzymology , Intellectual Disability/pathology , Mitochondria/enzymology , Mitochondria/pathology , Mitochondrial Diseases/enzymology , Mitochondrial Diseases/pathology , Mitochondrial Proteins/deficiency , Motor Neurons/cytology , Motor Neurons/metabolism , Muscle Weakness/enzymology , Muscle Weakness/pathology , Primary Cell Culture , Rhabdomyolysis/enzymology , Rhabdomyolysis/pathology , Ubiquinone/geneticsABSTRACT
We report the generation, CRISPR/Cas9-edition and characterization of induced pluripotent stem cell (iPSC) lines from a patient with coenzyme Q10 deficiency harboring the heterozygous mutation c.483G>C in the COQ4 gene. iPSCs were generated using non-integrative Sendai Viruses containing the reprogramming factors OCT4, SOX2, KLF4 and C-MYC. The iPSC lines carried the c.483G>C COQ4 mutation, silenced the OKSM expression and were mycoplasma-free. They were bona fide pluripotent cells as characterized by morphology, immunophenotype/gene expression for pluripotent-associated markers/genes, NANOG and OCT4 promoter demethylation, karyotype and teratoma formation. The COQ4 mutation was CRISPR/Cas9 edited resulting in isogenic, diploid and off-target free COQ4-corrected iPSCs.
Subject(s)
Genomics/methods , Induced Pluripotent Stem Cells/metabolism , Mitochondrial Proteins/genetics , Ubiquinone/analogs & derivatives , Cell Differentiation , Child, Preschool , Female , Heterozygote , Humans , Kruppel-Like Factor 4 , Mitochondrial Proteins/metabolism , Mutation , Ubiquinone/deficiencyABSTRACT
Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma.