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1.
Am J Psychiatry ; 179(5): 322-328, 2022 May.
Article En | MEDLINE | ID: mdl-35491564

There is an urgent and unmet need to advance our ability to translate genetic studies of psychiatric disorders into clinically actionable information, which could transform diagnostics and even one day lead to novel (and potentially presymptomatic) therapeutic interventions. Today, although there are hundreds of significant loci associated with psychiatric disorders, resolving the target gene(s) and pathway(s) impacted by each is a major challenge. Integrating human induced pluripotent stem cell-based approaches with CRISPR-mediated genomic engineering strategies makes it possible to study the impact of patient-specific variants within the cell types of the brain. As the scale and scope of functional genomic studies expands, so does our ability to resolve the complex interplay of the many risk variants linked to psychiatric disorders. In this review, the author discusses some of the technological advances that make it possible to ask exciting questions that are fundamental to our understanding of psychiatric disorders. How do distinct risk variants converge and interact with each other (and the environment) across the diverse cell types that comprise the human brain? Can clinical trajectories and/or therapeutic response be predicted from genetic profiles? Just as critically, by spreading the message that genetic risk for psychiatric disorders is biological and fundamentally no different than for other human conditions, we can dispel the stigma associated with mental illness.


Brain Diseases , Induced Pluripotent Stem Cells , Mental Disorders , Genome-Wide Association Study , Genomics , Humans , Induced Pluripotent Stem Cells/metabolism , Mental Disorders/genetics
2.
Methods Mol Biol ; 2429: 41-55, 2022.
Article En | MEDLINE | ID: mdl-35507154

Mouse somatic cell reprogramming using Oct4, Sox2, Klf4 and c-Myc (OSKM) induces formation of two stem cell types: induced pluripotent stem (iPS) cells and induced extraembryonic endoderm stem (iXEN) cells. Since both stem cells types routinely arise alongside one another during reprogramming, it is critical to distinguish between both cell types to ensure that the desired cell population is selected and analyzed. This chapter details, from start to finish, how to reprogram mouse embryonic fibroblasts (MEFs) using retrovirus and how to distinguish between iXEN and iPS cells at the colony and single-cell levels.


Induced Pluripotent Stem Cells , Pluripotent Stem Cells , Animals , Cell Differentiation/genetics , Cells, Cultured , Cellular Reprogramming/genetics , Endoderm/metabolism , Fibroblasts/metabolism , Induced Pluripotent Stem Cells/metabolism , Mice , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism
3.
Methods Mol Biol ; 2429: 73-84, 2022.
Article En | MEDLINE | ID: mdl-35507156

Protein aggregation is one of the hallmarks of many neurodegenerative diseases. While protein aggregation is a heavily studied aspect of neurodegenerative disease, methods of detection vary from one model system to another. Induced pluripotent stem cells (iPSCs) present an opportunity to model disease using patient-specific cells. However, iPSC-derived neurons are fetal-like in maturity, making it a challenge to detect key features such as protein aggregation that are often exacerbated with age. Nevertheless, we have previously found abnormal soluble and insoluble protein burden in motor neurons generated from amyotrophic lateral sclerosis (ALS) iPSCs, though protein aggregation has not been readily detected in iPSC-derived neurons from other neurodegenerative diseases. Therefore, here we present an ultracentrifugation method that detects insoluble protein species in various models of neurodegenerative disease, including Huntington's disease, Alzheimer's disease, and ALS. This method is able to detect soluble, insoluble, and SDS-resistant species in iPSC-derived neurons and is designed to be flexible for optimal detection of various aggregation-prone proteins.


Amyotrophic Lateral Sclerosis , Induced Pluripotent Stem Cells , Neurodegenerative Diseases , Amyotrophic Lateral Sclerosis/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Motor Neurons/metabolism , Neurodegenerative Diseases/metabolism , Protein Aggregates
4.
Cell Rep ; 39(5): 110790, 2022 05 03.
Article En | MEDLINE | ID: mdl-35508131

Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, cause a neurodevelopmental syndrome and increase the risk for schizophrenia. Using CRISPR-Cas9, we generate excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-). Our data show that SETD1A haploinsufficiency results in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype is primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling reveals perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identify specific changes in the cyclic AMP (cAMP)/Protein Kinase A pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, by pharmacologically targeting the cAMP pathway, we are able to rescue the network deficits in SETD1A+/- cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.


Induced Pluripotent Stem Cells , Schizophrenia , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Neurons/metabolism , Schizophrenia/genetics , Schizophrenia/metabolism
5.
Bioengineered ; 13(5): 11594-11601, 2022 May.
Article En | MEDLINE | ID: mdl-35510412

Previously, we demonstrated that the disheveled binding antagonist of ß-catenin 1 (DACT1) was involved in atrial fibrillation by regulating the reorganization of connexin 43 and ß-catenin in cardiomyocytes. Little is known, however, about DACT1 in human normal myocardial cells. Therefore, we used cardiomyocytes (CMs) derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to investigate the role of DACT1 and its connection with ß-catenin and connexin 43. While the ESC-CMs and iPSC-CMs were differentiated using commercial differentiation kits, the cardiac-specific markers were detected by immunofluorescence. The expression level of DACT1 was detected using western blotting, whereas the interaction of DACT1 and connexin 43 or ß-catenin was detected by immunofluorescence and co-immunoprecipitation (co-IP) assays. Both H1-CMs and SF-CMs were immunostained for cardiac-specific markers, including Troponin I, Troponin T, α-actinin, NKX2.5, and GATA6. While DACT1 was not expressed in both H1 ESCs and SF-iPSCs, it was, however, highly expressed in differentiated CMs, being also localized in the cytoplasm and the nucleus of differentiated CMs. Interestingly, the DACT1 expression in different nuclei was different in the same multinucleated cell. Moreover, DACT1 colocalized with ß-catenin in both the cytoplasm and nucleus of differentiated CMs, and it also colocalized with connexin 43 in the perinuclear region and the gap junctions of differentiated CMs. Co-IP results showed that DACT1 could directly bind to ß-catenin and connexin 43. Taken together, DACT1 interacted with ß-catenin and connexin 43 in human-induced pluripotent stem cells-derived cardiomyocytes.


Induced Pluripotent Stem Cells , Adaptor Proteins, Signal Transducing/metabolism , Cell Differentiation , Connexin 43/genetics , Connexin 43/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Nuclear Proteins/metabolism , beta Catenin/metabolism
6.
STAR Protoc ; 3(2): 101360, 2022 Jun 17.
Article En | MEDLINE | ID: mdl-35516845

Here we describe a protocol to obtain highly pure cardiomyocytes and neurons from human induced pluripotent stem cells (hiPSCs) via metabolic selection processes. Compared to conventional purification protocols, this approach is easier to perform and scale up and more cost-efficient. The protocol can be applied to hiPSCs and human embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Tohyama et al. (2016) and Tanosaki et al. (2020).


Induced Pluripotent Stem Cells , Pluripotent Stem Cells , Cell Differentiation/physiology , Fatty Acids/pharmacology , Humans , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/physiology , Neurons
7.
Stem Cells ; 40(1): 35-48, 2022 03 03.
Article En | MEDLINE | ID: mdl-35511867

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.


Histones , Induced Pluripotent Stem Cells , Animals , DNA , DNA Repair , Endothelial Cells/metabolism , Histones/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Mice , X-ray Repair Cross Complementing Protein 1/genetics , X-ray Repair Cross Complementing Protein 1/metabolism
8.
Stem Cell Res ; 61: 102766, 2022 May.
Article En | MEDLINE | ID: mdl-35367693

Schizophrenia is a chronic, serious and disabling mental disorder. Most patients can effectively control their condition through drug treatment, but there are still some patients who are difficult to gain benefits from drug treatment. Among them, the failure to respond to clozapine full-scale treatment is ultra-treatment-resistant schizophrenia. We generated induced pluripotent stem cells (iPSCs) from an ultra-treatment-resistant schizophrenia patient by electroporation of peripheral blood mononuclear cells (PBMCs) with episomal plasmids encoding OCT 4, SOX 2, NANOG, LIN 28, KLF 4 and MYC. The iPSCs demonstrated normal karyotype, expressed pluripotency markers and differentiated into the three germ layers in vivo.


Induced Pluripotent Stem Cells , Schizophrenia , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , Schizophrenia/genetics , Schizophrenia/metabolism , Transcription Factors/metabolism
9.
Stem Cell Res ; 61: 102765, 2022 May.
Article En | MEDLINE | ID: mdl-35378365

Mutations in PINK1 and Parkin are two of the main causes of recessive early-onset Parkinson's disease (PD). We generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of a 64-year-old male patient with a homozygous ILE368ASN mutation in PINK1, who experienced disease onset at 33 years, and from fibroblasts of a 61-year-old female patient heterozygous for the R275W mutation in Parkin, who experienced disease onset at 44 years. Array comparative genomic hybridization (aCGH) determined genotypic variation in each line. The cell lines were successfully used to generate midbrain dopaminergic neurons, the neuron type primarily affected in PD.


Induced Pluripotent Stem Cells , Parkinson Disease , Comparative Genomic Hybridization , Dopaminergic Neurons/metabolism , Female , Fibroblasts/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Male , Middle Aged , Mutation/genetics , Parkinson Disease/genetics , Parkinson Disease/metabolism , Protein Kinases/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
10.
Stem Cell Res ; 61: 102745, 2022 May.
Article En | MEDLINE | ID: mdl-35381520

Induced pluripotent stem cell (iPSC) line, SJHi001-A, was generated from a diabetic patient carrying a heterozygous c.G248A mutation in GCGR gene that generated the p.W83X. The PBMCs from her father (no diabetes and no mutation site) were also prepared into iPSC (SJHi002-A) as a control. Both two cell lines had normal karyotype, expressed pluripotency markers and could differentiate into the three germ layers in vivo.


Diabetes Mellitus , Induced Pluripotent Stem Cells , Cell Line , Diabetes Mellitus/genetics , Diabetes Mellitus/metabolism , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Mutation/genetics , Receptors, Glucagon/genetics , Receptors, Glucagon/metabolism
11.
Stem Cell Res ; 61: 102771, 2022 May.
Article En | MEDLINE | ID: mdl-35381521

Human-induced pluripotent stem cells (hiPSCs) clones NSi001-A, NSi001-B, and NSi001-C were generated from a female individual of Indian origin having Robertsonian translocation down syndrome (DS) by reprogramming peripheral blood mononuclear cells (PBMCs) using integration-free Sendai viral vectors. The established hiPSCs clones had karyotype similar to the patient sample with Robertsonian translocation [46, XX rob (14;21)], normal ES-like morphology, expression of pluripotency markers, and potential for three germ layer differentiation, i.e., ectoderm, mesoderm, and endoderm.


Down Syndrome , Induced Pluripotent Stem Cells , Cells, Cultured , Clone Cells , Down Syndrome/genetics , Down Syndrome/metabolism , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear , Translocation, Genetic
12.
Stem Cell Res ; 61: 102751, 2022 May.
Article En | MEDLINE | ID: mdl-35395622

Glomerulopathy with fibronectin deposits (GFND) is an autosomal dominant kidney disease exhibiting microscopic hematuria, proteinuria, and hypertension that may lead to end-stage renal failure. In this study, using non-integrative episomal vectors an induced pluripotent stem cell (iPSC) line, FHUSTCi001-A, was derived from peripheral blood mononuclear cells of an 11-year-old boy with GFND carrying a heterozygous c.5602G > A (p.V1868M) mutation in the FN1 gene. The generated iPSC line has a normal karyotype, expresses pluripotency markers, and has the capacity to form all three germ layers in vivo. This iPSC line offers a useful cellular model to study the pathogenesis of GFND disease.


Glomerulonephritis, Membranoproliferative , Induced Pluripotent Stem Cells , Child , Female , Glomerulonephritis, Membranoproliferative/metabolism , Glomerulonephritis, Membranoproliferative/pathology , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/pathology , Male , Mutation/genetics
13.
Stem Cell Res ; 61: 102776, 2022 May.
Article En | MEDLINE | ID: mdl-35397397

Oocyte maturation defect-4 (OOMD4) is an autosomal recessive disease characterized by oocyte maturation arrest. On chromosome 15q21, the PATL2 gene is mutated, resulting in OOMD4 in either a homozygous or compound heterozygous form. Herein, the peripheral blood mononuclear cells (PBMCs) were obtained from a female patient who was heterozygous for OOMD4 due to a PALT2 gene mutation. Then we obtained the induced pluripotent stem cell (iPSC) by using episomal vectors with transcription factors for reprogramming. The teratoma assay revealed that the iPSC line exhibited pluripotency with the ability to differentiate into three germ layers, namely ectoderm, mesoderm, and endoderm, with positive expression of their markers, such as TUJ, SMA, and AFP, respectively. Furthermore, a normal karyotype (46, XX) was observed. In this view, iPSCs can be a valuable tool for conducting extensive research on the OOMD4, establishing models, and identifying potential therapeutic targets.


Induced Pluripotent Stem Cells , Leukocytes, Mononuclear , Female , Heterozygote , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , Mutation/genetics , Oocytes
14.
Stem Cell Res ; 61: 102767, 2022 May.
Article En | MEDLINE | ID: mdl-35397398

In this study, we report the generation of a novel human induced pluripotent stem cell (hiPSC) line from bone marrow mononuclear cells of a patient with multiple myeloma, using an integrative Sendai virus vector. This pluripotent cell line has been shown to differentiate into three germ layers. Therefore, these induced pluripotent stem cells (iPSCs) will enable not only advances in cell therapy products but also the study of mechanisms.


Induced Pluripotent Stem Cells , Multiple Myeloma , Cell Line , Germ Layers , Humans , Induced Pluripotent Stem Cells/metabolism , Multiple Myeloma/metabolism , Sendai virus/genetics
15.
Stem Cell Res ; 61: 102777, 2022 May.
Article En | MEDLINE | ID: mdl-35405382

We generated a human induced pluripotent stem cell (iPSC) line, FMUPDCi001-A, from peripheral blood mononuclear cells of a patient with mental retardation, autosomal recessive 36 (MRT36), and compound heterozygous c.219dupA and c.587C > T variants in ADAT3. This line will be a valuable resource for investigating disease mechanisms and testing gene therapies for MRT36.


Induced Pluripotent Stem Cells , Intellectual Disability , Humans , Induced Pluripotent Stem Cells/metabolism , Intellectual Disability/genetics , Intellectual Disability/metabolism , Leukocytes, Mononuclear
16.
Stem Cell Res ; 61: 102783, 2022 May.
Article En | MEDLINE | ID: mdl-35413565

Otosclerosis is caused by abnormal bone remodeling in the middle ear, resulting in progressive hearing loss, dizziness, balance problems, and tinnitus. Previous infection, stress fractures of the bony tissue surrounding the inner ear, immune disorders, and genetic factors are believed to contribute to this disease. Currently, no effective drug treatment for otosclerosis is known. Herein, we generated an induced pluripotent stem cell line from the peripheral blood mononuclear cells of an otosclerosis patient. The cell line exhibited normal morphology, karyotype, and pluripotency marker expression. A teratoma assay revealed successful differentiation into all three germ layers.


Induced Pluripotent Stem Cells , Otosclerosis , Teratoma , Cell Line , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear , Otosclerosis/metabolism , Teratoma/metabolism
17.
Stem Cell Res ; 61: 102774, 2022 May.
Article En | MEDLINE | ID: mdl-35413566

Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disorder characterized by a thick left ventricular wall and an increased risk of arrhythmias, heart failure, and sudden cardiac death. The MYBPC3 and PRAKG2 are known causal genes for HCM. Here we generated two human-induced pluripotent stem cell lines from two HCM patients carrying two heterozygous mutations in MYBPC3 (c.459delC) and PRKAG2 (c.1703C > T). Both iPSC lines expressed pluripotent markers, had a normal karyotype, and were able to differentiate into three germ layers, making them potentially valuable tools for modeling HCM in vitro and investigating the pathological mechanisms related to these two variants.


Cardiomyopathy, Hypertrophic , Induced Pluripotent Stem Cells , AMP-Activated Protein Kinases/genetics , Cardiomyopathy, Hypertrophic/pathology , Cytoskeletal Proteins/genetics , Heterozygote , Humans , Induced Pluripotent Stem Cells/metabolism , Mutation
18.
Stem Cell Res ; 61: 102775, 2022 May.
Article En | MEDLINE | ID: mdl-35413567

Combined Oxidative Phosphorylation Deficiency 23 (COXPD23) caused by mutations in GTPBP3 gene is a rare mitochondrial disease. The patient-derived PBMCs of sibling with the compound heterozygous variants in GTPBP3 (NM_133644): c.1289G>A(p.Cys430Tyr); c.545G>A(p.Gly182Glu) were reprogrammed into induced pluripotent stemcell (iPSC) lines (DPNJMUi001-A.) using non-integrative Sendai virus. The COXPD23 iPSC lines present normal karyotypes, high expression of pluripotency markers and the capacity to differentiate into cells of all three germ layers.


Induced Pluripotent Stem Cells , Female , GTP-Binding Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , Mutation/genetics , Sendai virus , Siblings
19.
Stem Cell Res ; 61: 102782, 2022 May.
Article En | MEDLINE | ID: mdl-35421843

Spinal cerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is the result of abnormal repeat amplification of CAG of the ATXN3 gene. It is one of the main types of autosomal dominant ataxia, with motor symptoms of cerebellar ataxia, mainly accompanied by non-motor symptoms, such as ocular symptoms, psychiatric symptoms, and nutritional disorders. Currently, no effective treatment is available for patients with SCA3. The construction of induced pluripotent stem cells (iPSCs) from two SCA3 patients (14/74 CAG repeats) will be an excellent tool for studying SCA3 disease mechanisms and for drug screening.


Cerebellar Ataxia , Induced Pluripotent Stem Cells , Ataxin-3/genetics , Ataxin-3/metabolism , Cerebellar Ataxia/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , Mutation/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism
20.
Stem Cell Res ; 61: 102768, 2022 May.
Article En | MEDLINE | ID: mdl-35421845

Epilepsy is a common chronic neurological disorder related to genetic factors. Base on the non-integrating episomal vector technique, a human induced pluripotent stem cell (iPSC) line, termed as LZUSHI001-A, was generated from peripheral blood mononuclear cells (PBMCs) of a 11-year-old male patient with Epilepsy, who had a heterozygous (c.2042G>A, p.R681Q) mutation in the DGKG gene. LZUSHI001-A offers a useful resource to investigate pathogenic mechanisms in epilepsy, as well as a cell-based model for drug development to treat epilepsy.


Epilepsy , Induced Pluripotent Stem Cells , Child , Epilepsy/genetics , Epilepsy/pathology , Heterozygote , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear , Male , Mutation/genetics
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