scholarly journals Evaluation of ATG5 Gene Expression in Human Induced Pluripotent Stem Cells During Endoderm Induction

2020 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Alice Sabet ◽  
Negar Azarpira ◽  
Saeid Ghavami ◽  
Leila Kohan
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Mrna Expression ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Metabolic Stress ◽  
High Rate ◽  
Human Ipscs ◽  
Induced Pluripotent

Background: Autophagy is a vital cell survival mechanism that authorizes cells to assort to metabolic stress and is essential for the development and maintenance of cellular and tissue homeostasis, as well as the prevention of human disease. It has also been shown that autophagy plays a significant role in the development and differentiation of stem cells, as well as induced pluripotent stem cells (iPSCs). Objectives: The present study aimed to examine the mRNA expression of the ATG5 gene, one of the key markers of autophagy in human iPSCs (hiPSCs) during endoderm induction. Methods: In this study, we cultured the human iPSC line (R1-hiPSC1) on mitomycin-C, inactivated mouse embryonic fibroblasts (MEF) layer, and used hanging drop protocol to generate embryoid body (EB) and expose differentiation. The Real-time PCR method was used to examine the mRNA expression level of ATG5 in hiPSC during endoderm induction. Results: Our results demonstrated the high mRNA expression of ATG5 in the MEI stage, which shows the high rate of autophagy in MEI days rather than the other stages of differentiation. Conclusions: The modification of ATG5 gene expression within hiPSC during endoderm induction shows the importance of autophagy assessments in hiPSC differentiation. Therefore, subsequent studies are needed to clarify the details of autophagy effects on hiPSC differentiation.

scholarly journals Improving the efficiency of gene insertion in a human artificial chromosome vector and its transfer in human-induced pluripotent stem cells

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Yoshinori Hasegawa ◽  
Masashi Ikeno ◽  
Nobutaka Suzuki ◽  
Manabu Nakayama ◽  
Osamu Ohara
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Artificial Chromosome ◽  
Human Artificial Chromosome ◽  
Gene Insertion ◽  
Size Limitation ◽  
Human Ipscs ◽  
Induced Pluripotent

Abstract A human artificial chromosome (HAC) vector has potential to overcome the problems of stable gene expression associated with plasmid, transposon, and virus-based vectors, such as insertional mutagenesis, position effect, uncontrollable copy number, unstable gene expression, and DNA size limitation. The main advantages of the HAC are its episomal nature and ability to accommodate DNA inserts of any size. However, HAC vectors have two disadvantages: low efficiency of gene insertion and lack of reports regarding the successful HAC transfer to human-induced pluripotent stem cells (iPSCs). We here provide the first report of a method for the efficient transfer of HAC to human iPSCs for obtaining reproducible experimental results. Moreover, we achieved a 10% increase in the gene insertion efficiency in the HAC vector using our new site-specific recombination systems VCre/VloxP and SCre/SloxP.

scholarly journals Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia

2021 ◽  
Vol 22 (9) ◽  
pp. 4334
Author(s):  
Katrina Albert ◽  
Jonna Niskanen ◽  
Sara Kälvälä ◽  
Šárka Lehtonen
Keyword(s):  
Stem Cells ◽  
Neurodegenerative Diseases ◽  
Neurodegenerative Disease ◽  
Induced Pluripotent Stem Cells ◽  
Glial Cells ◽  
Pluripotent Stem Cells ◽  
Cell Types ◽  
Human Ipscs ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

scholarly journals A non-invasive method to generate induced pluripotent stem cells from primate urine

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Johanna Geuder ◽  
Lucas E. Wange ◽  
Aleksandar Janjic ◽  
Jessica Radmer ◽  
Philipp Janssen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Sendai Virus ◽  
Cell Types ◽  
Urine Samples ◽  
Comparative Approach ◽  
Non Invasive ◽  
Human Ipscs ◽  
Induced Pluripotent

AbstractComparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.

scholarly journals T9. EPIGENETIC PROFILING IN SCHIZOPHRENIA DERIVED HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) AND NEURONS

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S234-S234
Author(s):  
Lorna Farrelly ◽  
Shuangping Zhang ◽  
Erin Flaherty ◽  
Aaron Topol ◽  
Nadine Schrode ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Neural Progenitor Cells ◽  
Gene Expression Pattern ◽  
Early Gene ◽  
Label Free ◽  
Induced Pluripotent

Abstract Background Schizophrenia (SCZ) is a severe psychiatric disorder affecting ~1% of the world’s population. It is largely heritable with genetic risk reflected by a combination of common variants of small effect and highly penetrant rare mutations. Chromatin modifications are known to play critical roles in the mediation of many neurodevelopmental processes, and, when disturbed, may also contribute to the precipitation of psychiatric disorders, such as SCZ. While a handful of candidate-based studies have measured changes in promoter-bound histone modifications, few mechanistic studies have been carried out to explore how these modifications may affect chromatin to precipitate behavioral phenotypes associated with the disease. Methods We applied an unbiased proteomics approach to evaluate the epigenetic landscape of SCZ in human induced pluripotent stem cells (hiPSC), neural progenitor cells (NPCs) and neurons from SCZ patients vs. matched controls. We utilized proteomics-based, label free liquid chromatography mass spectrometry (LC-MS/MS) on purified histones from these cells and confirmed our results by western blotting in postmortem SCZ cortical brain tissues. Furthermore we validated our findings with the application of histone interaction assays and structural and biophysical assessments to identify and confirm novel chromatin ‘readers’. To relate our findings to a SCZ phenotype we used a SCZ rodent model of prepulse inhibition (PPI) to perform pharmacological manipulations and behavioral assessments. Results Using label free mass spectrometry we performed PTM screening of hiPSCs, NPCs and matured neurons derived from SCZ patients and matched controls. We identified, amongst others, altered patterns of hyperacetylation in SCZ neurons. Additionally we identified enhanced binding of particular acetylation ‘reader’ proteins. Pharmacological inhibition of such proteins in an animal model of amphetamine sensitization ameliorated PPI deficits further validating this epigenetic signature in SCZ. Discussion Recent evidence indicates that relevance and patterns of acetylation in epigenetics advances beyond its role in transcription and small molecule inhibitors of these aberrant interactions hold promise as useful therapeutics. This study identifies a role for modulating gene expression changes associated with a SCZ epigenetic signature and warrants further investigation in terms of how this early gene expression pattern perhaps determines susceptibility or severity of the SCZ disease trajectory.

Effective embryoid body formation from induced pluripotent stem cells for regeneration of respiratory epithelium

2013 ◽  
Vol 124 (1) ◽  
pp. E8-E14 ◽  
Author(s):  
Koshi Otsuki ◽  
Mitsuyoshi Imaizumi ◽  
Yukio Nomoto ◽  
Mika Nomoto ◽  
Ikuo Wada ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Respiratory Epithelium ◽  
Body Formation ◽  
Induced Pluripotent ◽  
Embryoid Body Formation

scholarly journals Alterations of Glycosphingolipid Glycans and Chondrogenic Markers during Differentiation of Human Induced Pluripotent Stem Cells into Chondrocytes

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1622
Author(s):  
Liang Xu ◽  
Hisatoshi Hanamatsu ◽  
Kentaro Homan ◽  
Tomohiro Onodera ◽  
Takuji Miyazaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Chondrogenic Differentiation ◽  
Cell Types ◽  
Human Cartilage ◽  
Human Ipscs ◽  
Normal Human ◽  
Induced Pluripotent ◽  
Promising Source

Due to the limited intrinsic healing potential of cartilage, injury to this tissue may lead to osteoarthritis. Human induced pluripotent stem cells (iPSCs), which can be differentiated into chondrocytes, are a promising source of cells for cartilage regenerative therapy. Currently, however, the methods for evaluating chondrogenic differentiation of iPSCs are very limited; the main techniques are based on the detection of chondrogenic genes and histological analysis of the extracellular matrix. The cell surface is coated with glycocalyx, a layer of glycoconjugates including glycosphingolipids (GSLs) and glycoproteins. The glycans in glycoconjugates play important roles in biological events, and their expression and structure vary widely depending on cell types and conditions. In this study, we performed a quantitative GSL-glycan analysis of human iPSCs, iPSC-derived mesenchymal stem cell like cells (iPS-MSC like cells), iPS-MSC-derived chondrocytes (iPS-MSC-CDs), bone marrow-derived mesenchymal stem cells (BMSCs), and BMSC-derived chondrocytes (BMSC-CDs) using glycoblotting technology. We found that GSL-glycan profiles differed among cell types, and that the GSL-glycome underwent a characteristic alteration during the process of chondrogenic differentiation. Furthermore, we analyzed the GSL-glycome of normal human cartilage and found that it was quite similar to that of iPS-MSC-CDs. This is the first study to evaluate GSL-glycan structures on human iPS-derived cartilaginous particles under micromass culture conditions and those of normal human cartilage. Our results indicate that GSL-glycome analysis is useful for evaluating target cell differentiation and can thus support safe regenerative medicine.

scholarly journals Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Laís Vicari de Figueiredo Pessôa ◽  
Pedro Ratto Lisboa Pires ◽  
Maite del Collado ◽  
Naira Caroline Godoy Pieri ◽  
Kaiana Recchia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Mirna Profile ◽  
Patient Specific ◽  
Induced Pluripotent

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

scholarly journals Preferential gene expression and epigenetic memory of induced pluripotent stem cells derived from mouse pancreas

2015 ◽  
Vol 20 (5) ◽  
pp. 367-381 ◽  
Author(s):  
Daiki Nukaya ◽  
Kohtaro Minami ◽  
Ritsuko Hoshikawa ◽  
Norihide Yokoi ◽  
Susumu Seino
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Epigenetic Memory ◽  
Mouse Pancreas ◽  
Induced Pluripotent

scholarly journals Derivation of induced pluripotent stem cells from ferret somatic cells

2020 ◽  
Vol 318 (4) ◽  
pp. L671-L683
Author(s):  
Jinghui Gao ◽  
Sophia Petraki ◽  
Xingshen Sun ◽  
Leonard A. Brooks ◽  
Thomas J. Lynch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Preclinical Model ◽  
Specific Cell ◽  
Chronic Lung Diseases ◽  
Human Ipscs ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.

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