induced pluripotent stem cells
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2022 ◽  
Vol 164 ◽  
pp. 136-147
Author(s):  
Giulia Emanuelli ◽  
Anna Zoccarato ◽  
Christina M. Reumiller ◽  
Angelos Papadopoulos ◽  
Mei Chong ◽  
...  

2022 ◽  
Author(s):  
Tuyana Malankhanova ◽  
Dayaana Vasileva ◽  
Elena Grigor'eva ◽  
Sergey Medvedev ◽  
Suren Zakian ◽  
...  

Many neurodegenerative diseases, including Huntington’s disease (HD), are associated with oxidative stress in the neurons of the brain. Genetically encoded biosensorsare useful for studying these processesin vitro. Human cell cultures expressing the biosensors can serve as a cell model for developing and testing effective agents that reduce oxidative stress. In this work, transgenes encoding biosensors of glutathione oxidative potential(Grx1-roGFP2) with cytoplasmic and mitochondrial localization were introduced into human induced pluripotent stem cells of a healthy donor and an HD patient using CRISPR/Cas9-mediated genome editing. The cells were subsequently differentiated into medium spiny neurons of the striatum. The expression of the biosensors was detected in the iPSCs, neuronal precursors and mature neurons.The obtained cells could be used to study the redox potential of glutathione in HD neurons and to screen for new drug compounds aimed at reducing oxidative stress. Keywords: genetically encoded biosensors, Huntington’s disease, induced pluripotent stem cells, medium spiny neurons, oxidative stress, glutathione, Grx1-roGFP2


2022 ◽  
Vol 8 ◽  
Author(s):  
Warunya Chakritbudsabong ◽  
Ladawan Sariya ◽  
Phakhin Jantahiran ◽  
Nattarun Chaisilp ◽  
Somjit Chaiwattanarungruengpaisan ◽  
...  

The reprogramming of cells into induced neural stem cells (iNSCs), which are faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for fundamental and frontier research, as well as personalized cell-based therapies for neurological diseases. However, reprogramming cells with viral vectors increases the risk of tumor development due to vector and transgene integration in the host cell genome. To circumvent this issue, the Sendai virus (SeV) provides an alternative integration-free reprogramming method that removes the danger of genetic alterations and enhances the prospects of iNSCs from bench to bedside. Since pigs are among the most successful large animal models in biomedical research, porcine iNSCs (piNSCs) may serve as a disease model for both veterinary and human medicine. Here, we report the successful generation of piNSC lines from pig fibroblasts by employing the SeV. These piNSCs can be expanded for up to 40 passages in a monolayer culture and produce neurospheres in a suspension culture. These piNSCs express high levels of NSC markers (PAX6, SOX2, NESTIN, and VIMENTIN) and proliferation markers (KI67) using quantitative immunostaining and western blot analysis. Furthermore, piNSCs are multipotent, as they are capable of producing neurons and glia, as demonstrated by their expressions of TUJ1, MAP2, TH, MBP, and GFAP proteins. During the reprogramming of piNSCs with the SeV, no induced pluripotent stem cells developed, and the established piNSCs did not express OCT4, NANOG, and SSEA1. Hence, the use of the SeV can reprogram porcine somatic cells without first going through an intermediate pluripotent state. Our research produced piNSCs using SeV methods in novel, easily accessible large animal cell culture models for evaluating the efficacy of iNSC-based clinical translation in human medicine. Additionally, our piNSCs are potentially applicable in disease modeling in pigs and regenerative therapies in veterinary medicine.


BMC Biology ◽  
2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Joffrey Mianné ◽  
Amel Nasri ◽  
Chloé Nguyen Van ◽  
Chloé Bourguignon ◽  
Mathieu Fieldès ◽  
...  

Abstract Background The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. Results We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. Conclusions Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.


2022 ◽  
Author(s):  
Hajime Ozaki ◽  
Hidetaka Suga ◽  
Mayu Sakakibara ◽  
Mika Soen ◽  
Natsuki Miyake ◽  
...  

Abstract Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs) differentiate into AVP neurons, whereas human ESCs/induced pluripotent stem cells (iPSCs) die. Human ES/iPSCs are generally more similar to mouse epiblast stem cells compared to mouse ESCs, which are termed as primed and naive, respectively. In this study, we converted human FNDI-specific iPSCs from primed to naive cells, and found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.


2022 ◽  
Vol 23 (2) ◽  
pp. 623
Author(s):  
Junya Zhao ◽  
Miaojin Zhou ◽  
Zujia Wang ◽  
Lingqian Wu ◽  
Zhiqing Hu ◽  
...  

Hemophilia A (HA) is caused by mutations in the coagulation factor VIII (FVIII) gene (F8). Gene therapy is a hopeful cure for HA; however, FVIII inhibitors formation hinders its clinical application. Given that platelets promote coagulation via locally releasing α-granule, FVIII ectopically expressed in platelets has been attempted, with promising results for HA treatment. The B-domain-deleted F8 (BDDF8), driven by a truncated ITGA2B promoter, was targeted at the ribosomal DNA (rDNA) locus of HA patient-specific induced pluripotent stem cells (HA-iPSCs). The F8-modified, human induced pluripotent stem cells (2bF8-iPSCs) were differentiated into induced hematopoietic progenitor cells (iHPCs), induced megakaryocytes (iMKs), and mesenchymal stem cells (iMSCs), and the FVIII expression was detected. The ITGA2B promoter-driven BDDF8 was site-specifically integrated into the rDNA locus of HA-iPSCs. The 2bF8-iPSCs were efficiently differentiated into 2bF8-iHPCs, 2bF8-iMKs, and 2bF8-iMSCs. FVIII was 10.31 ng/106 cells in lysates of 2bF8-iHPCs, compared to 1.56 ng/106 cells in HA-iHPCs, and FVIII was 3.64 ng/106 cells in 2bF8-iMSCs lysates, while 1.31 ng/106 cells in iMSCs with CMV-driven BDDF8. Our results demonstrated a high expression of FVIII in iHPCs and iMSCs derived from hiPSCs with site-specific integration of ITGA2B promoter-driven BDDF8, indicating potential clinical prospects of this platelet-targeted strategy for HA gene therapy.


2022 ◽  
Vol 14 (1) ◽  
Author(s):  
Jeeranan Manokawinchoke ◽  
Phoonsuk Limraksasin ◽  
Hiroko Okawa ◽  
Prasit Pavasant ◽  
Hiroshi Egusa ◽  
...  

AbstractIn vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.


Author(s):  
Fumitaka Obayashi ◽  
Atsuko Hamada ◽  
Sachiko Yamasaki ◽  
Taku Kanda ◽  
Shigeaki Toratani ◽  
...  

AbstractCowden syndrome (CS) is an autosomal dominant inherited disorder characterized by multiple hamartomas in various organs such as the mucosa, skin, and gastrointestinal tract. Patients with CS are at high risk for breast and thyroid cancers. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that negatively regulates the AKT pathway, and PTEN mutations are known to be the major causes of this syndrome. However, the pathogenesis of this syndrome has not been clarified. Here, we present a case of a Japanese woman with multiple oral polyps, breast cancer, and thyroid cancer who was clinically diagnosed with CS. We obtained DNA and RNA samples from the patient’s peripheral blood mononuclear cells (PBMCs) and buccal mucosa tumor. Next-generation sequencing revealed novel germline mutations (c.1020delT and c.1026G > A) in exon 8 of PTEN. Sanger sequencing identified no PTEN transcript from the mutant allele. Furthermore, CS-specific induced pluripotent stem cells (CS-iPSCs) were established from PBMCs of the patient under feeder- and serum-free culture. Compared with healthy PBMCs and iPSCs, both of the CS-derived PBMCs and CS-iPSCs exhibited significantly reduced expression of the PTEN transcript. The transcriptional variant, PTENδ, was increased in CS-iPSCs, suggesting that it may be the cause of the disease.


2022 ◽  
Author(s):  
Jian Xu ◽  
Rui Wang ◽  
Jingxin Zhao ◽  
Fumei He ◽  
Yidi Zhang ◽  
...  

Abstract Background: Transcription factors (TFs) have been introduced to drive highly efficient differentiation of human induced pluripotent stem cells (hiPSCs) into lineage-specific oligodendrocytes (OLs). However, effective strategies currently rely mainly on genome-integrating viruses. To facilitate the translation of hiPSC-derived OLs into clinical practice, a synthetic modified messenger RNA (smRNA) reprogramming method that generates transgene-free OLs has been developed.Methods: An smRNA encoding Olig2, a key TF in OL development, with a defined phosphorylation site modification serine 147 replaced with alanine, Olig2S147A, was designed to reprogram hiPSCs into OLs. Proteomics were used to identify Olig2S147A-binding proteins that positively mediated of Olig2S147A-driven OL differentiation.Results: We demonstrated that repeated administration of the smRNA encoding Olig2 S147A led to higher and more stable protein expression. Using the single-mutant Olig2 smRNA with morphogens, we established a 6-day smRNA transfection protocol, and glial induction led to rapid NG2+ OL progenitor cell (OPC) generation (>70% purity) from hiPSC-derived neural progenitor cells (NPCs). The smRNA-induced NG2+ OPCs matured into functional OLs and myelinated nanofibers in vitro. Moreover, when transplanted into mice with cuprizone-induced demyelination, smRNA-induced OPCs promoted remyelination ex vivo. A proteomic analysis of Olig2-binding proteins indicated that the heat shock protein 70 (HSP70) complex bound Olig2. The HSP70 complex bound more strongly to Olig2 with the modified phosphorylation site than to wild type Olig2. VER-155008, an HSP70 complex antagonist, and ML346, an HSP70 complex agonist, inhibited and promoted Olig2 transcriptional activity and efficient OL generation, respectively.Conclusions: We present a very safe and efficient smRNA-driven strategy for hiPSC differentiation into OLs, which might be utilized for disease modeling, drug discovery, and/or therapeutic OPC/OL transplantation in neurodegenerative disease.


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