scholarly journals Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors

2018 ◽  
Author(s):  
Koichiro Nishino ◽  
Yoshikazu Arai ◽  
Ken Takasawa ◽  
Masashi Toyoda ◽  
Mayu Yamazaki-Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Delivery ◽  
Sendai Virus ◽  
Embryonic Stem ◽  
Aberrant Methylation ◽  
Delivery Methods ◽  
Genome Wide ◽  
Human Ipscs ◽  
Episomal Vectors ◽  
Induced Pluripotent

AbstractHuman induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hyper-methylated regions compared with embryonic stem cells. These random aberrant hyper-methylations might be a cause of the differences in the properties of each of the iPSC lines.

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 Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 261 ◽  
Author(s):  
Sergey Sinenko ◽  
Elena Skvortsova ◽  
Mikhail Liskovykh ◽  
Sergey Ponomartsev ◽  
Andrey Kuzmin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Biomedical Applications ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
Human Artificial Chromosome ◽  
Human Escs ◽  
Human Ipscs ◽  
Induced Pluripotent

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.

Cardiomyocyte differentiation of pluripotent stem cells and their use as cardiac disease models

2011 ◽  
Vol 434 (1) ◽  
pp. 25-35 ◽  
Author(s):  
Cheryl Dambrot ◽  
Robert Passier ◽  
Douwe Atsma ◽  
Christine L. Mummery
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cardiac Disease ◽  
Coming Of Age ◽  
Embryonic Stem ◽  
Daily Basis ◽  
Human Cardiomyocytes ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
Exciting Area

More than 10 years after their first isolation, human embryonic stem cells are finally ‘coming of age’ in research and biotechnology applications as protocols for their differentiation and undifferentiated expansion in culture become robust and scalable, and validated commercial reagents become available. Production of human cardiomyocytes is now feasible on a daily basis for many laboratories with tissue culture expertise. An additional recent surge of interest resulting from the first production of human iPSCs (induced pluripotent stem cells) from somatic cells of patients now makes these technologies of even greater importance since it is likely that (genetic) cardiac disease phenotypes can be captured in the cardiac derivatives of these cells. Although cell therapy based on replacing cardiomyocytes lost or dysfunctional owing to cardiac disease are probably as far away as ever, biotechnology and pharmaceutical applications in safety pharmacology and drug discovery will probably impact this clinical area in the very near future. In the present paper, we review the cutting edge of this exciting area of translational research.

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

2020 ◽  
Author(s):  
Johanna Geuder ◽  
Mari Ohnuki ◽  
Lucas E. Wange ◽  
Aleksandar Janjic ◽  
Johannes W. Bagnoli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Rna Sequencing ◽  
Human Urine ◽  
Pluripotent Stem Cells ◽  
Sendai Virus ◽  
Culture Conditions ◽  
Non Invasive ◽  
Human Ipscs ◽  
Induced Pluripotent

SummaryComparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically even 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 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 generate iPSCs non-invasively from primate urine. This will allow to extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.Graphical AbstractWorkflow overview for establishing iPSCs from primate urine(A) We established the protocol for human urine based on a previous description (Zhou 2012). We tested volume, storage and culture conditions for primary cells and compared reprogramming by overexpression of OCT3/4, SOX2, KLF4 and MYC (OSKM) via lipofection of episomal vectors and via transduction of a sendai virus derived vector (SeV). (B) We used the the protocol established in humans and adapted it for unsterile floor-collected samples from non-human primates by adding Normocure to the first passages of primary cell culture and reprogrammed visually healthy and uncontaminated cultures using SeV. (C) Pluripotency of established cultures was verified by marker expression, differentiation capacity and cell type classification using RNA sequencing.

Role of SOX17 in hematopoietic development from human embryonic stem cells

Blood ◽  
2013 ◽  
Vol 121 (3) ◽  
pp. 447-458 ◽  
Author(s):  
Yaeko Nakajima-Takagi ◽  
Mitsujiro Osawa ◽  
Motohiko Oshima ◽  
Haruna Takagi ◽  
Satoru Miyagi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Global Gene Expression ◽  
Gene Encoding ◽  
Hematopoietic Development ◽  
Genome Wide ◽  
Regulator Genes ◽  
Induced Pluripotent

Abstract To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34+CD43− endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested, only Sox17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34+CD43+CD45−/low cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34+CD43− ECs compared with low levels in CD34+CD43+CD45− pre-hematopoietic progenitor cells (pre-HPCs) and CD34+CD43+CD45+ HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34+CD43+CD45−/low cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation directly. Depletion of SOX17 in CD34+CD43− ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs, thereby regulating hematopoietic development from hESCs/iPSCs.

scholarly journals Electrophysiological properties of human induced pluripotent stem cells

2010 ◽  
Vol 298 (3) ◽  
pp. C486-C495 ◽  
Author(s):  
Peng Jiang ◽  
Stephanie N. Rushing ◽  
Chi-wing Kong ◽  
Jidong Fu ◽  
Deborah Kuo-Ti Lieu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Delayed Rectifier ◽  
Channel Blockers ◽  
Voltage Gated ◽  
Human Ipscs ◽  
Induced Pluripotent

Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to induced pluripotent stem cells (iPSCs) has been reported. Although hESCs and human iPSCs have been shown to share a number of similarities, such basic properties as the electrophysiology of iPSCs have not been explored. Previously, we reported that several specialized ion channels are functionally expressed in hESCs. Using transcriptomic analyses as a guide, we observed tetraethylammonium (TEA)-sensitive (IC50 = 3.3 ± 2.7 mM) delayed rectifier K+ currents ( IKDR) in 105 of 110 single iPSCs (15.4 ± 0.9 pF). IKDR in iPSCs displayed a current density of 7.6 ± 3.8 pA/pF at +40 mV. The voltage for 50% activation ( V1/2) was −7.9 ± 2.0 mV, slope factor k = 9.1 ± 1.5. However, Ca2+-activated K+ current ( IKCa), hyperpolarization-activated pacemaker current ( If), and voltage-gated sodium channel (NaV) and voltage-gated calcium channel (CaV) currents could not be measured. TEA inhibited iPSC proliferation (EC50 = 7.8 ± 1.2 mM) and viability (EC50 = 5.5 ± 1.0 mM). By contrast, 4-aminopyridine (4-AP) inhibited viability (EC50 = 4.5 ± 0.5 mM) but had less effect on proliferation (EC50 = 0.9 ± 0.5 mM). Cell cycle analysis further revealed that K+ channel blockers inhibited proliferation primarily by arresting the mitotic phase. TEA and 4-AP had no effect on iPSC differentiation as gauged by ability to form embryoid bodies and expression of germ layer markers after induction of differentiation. Neither iberiotoxin nor apamin had any function effects, consistent with the lack of IKCa in iPSCs. Our results reveal further differences and similarities between human iPSCs and hESCs. A better understanding of the basic biology of iPSCs may facilitate their ultimate clinical application.

scholarly journals KDM6A knockout in human iPSCs alters the genome-wide histone methylation profile at active and poised enhancers, activating expression of ectoderm gene expression pathways

2021 ◽  
Author(s):  
Shailendra S. Maurya ◽  
Wei Yang ◽  
Qiang Zhang ◽  
Petra Erdmann-Gilmore ◽  
Amelia Bystry ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Histone Modification ◽  
Histone Modifications ◽  
Embryonic Stem ◽  
Specific Gene ◽  
Regulatory Domains ◽  
Genome Wide ◽  
Human Ipscs ◽  
Expression Pathways

AbstractKDM6A is a histone demethylase, known to remove methyl moieties at the lysine residues of histone 3-labeled (H3K27me3) poised enhancers and bivalent promoters, which regulates gene expression during the differentiation of embryonic stem cells and tissue-specific development. However, while tissue- and disease-specific analyses have been performed, little is known about the location and consequences on gene expression of these regulatory regions in human pluripotent cells. Poised enhancers and bivalent promoters function in a coordinated fashion during development, which requires timely and efficient histone modifications. Identification of KDM6A-specific gene-regulatory domains is important for understanding the developmental mechanisms controlled by these histone modifications in pluripotency. In this study, we compared genome-wide histone modification and gene expression differences in isogenic wild type and cas9-mediated KDM6A knockout human induced pluripotent stem cells (hiPSC) lines. Here, we report that the absence of KDM6A does not alter the pluripotent phenotype but does substantially alter the histone modification profile at poised and active enhancers, resulting in decreased expression of associated COMPASS complex genes KMT2C and KMT2D and subsequently increasing the expression of gene pathways involved in ectoderm differentiation.

13. A Novel Method of Culturing Murine Induced Pluripotent Stem Cells (iPSCs)

2016 ◽  
Author(s):  
Kirsten Sjonnesen
Keyword(s):  
Stem Cells ◽  
Large Scale ◽  
Embryonic Stem ◽  
Cell Lineages ◽  
Human Ipscs ◽  
Novel Method ◽  
Vitro Model ◽  
Induced Pluripotent

Similar to embryonic stem cells (ESCs), iPSCs have the ability to differentiate into all three cell lineages, while not being subjected to the ethical complications attributed to ESCs. Our project goal was to develop a novel method of culturing murine iPSCs, reprogrammed from mouse embryonic fibroblasts, in large-scale quantities while maintaining their pluripotent characteristics and genomic integrity over a long term period. Stirred Suspension Bioreactors (SSBs) propose several benefits over static culture systems and facilitate the large-scale, economical expansion required for clinical studies. In our work with the SSB system, various techniques were used to promote and analyze the continued pluripotency of the murine iPSCs. Our results showed that iPSCs maintained their pluripotent state in SSBs and retained their ability to differentiate into different cell lineages after long term culture. miPSC were induced to differentiate into heart, bone and cartilage tissues at the end of maintenance period. The cumulative cell-fold expansion of iPSCs in suspension culture was 8.8×1010 cells, with 75% mean pluripotency at each passage. The cells expressed the major pluripotency markers as detected by RT-PCR. This study confirms the utility of the SSB system as a tool to cultivate large quantities of functional mouse iPSCs. SSBs have tremendous potential in future clinical applications and work has now begun to applying this system towards culturing human iPSCs. One potential application is the derivation of disease and patient derived iPSCs, which can recapitulate the disease phenotype for use as an in vitro model.

scholarly journals Disruption of RING and PHD Domains of TRIM28 Evokes Differentiation in Human iPSCs

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1933
Author(s):  
Sylwia Mazurek ◽  
Urszula Oleksiewicz ◽  
Patrycja Czerwińska ◽  
Joanna Wróblewska ◽  
Marta Klimczak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Human Ipscs ◽  
New Gene ◽  
Induced Pluripotent ◽  
The Impact ◽  
Phd Domain

TRIM28, a multi-domain protein, is crucial in the development of mouse embryos and the maintenance of embryonic stem cells’ (ESC) self-renewal potential. As the epigenetic factor modulating chromatin structure, TRIM28 regulates the expression of numerous genes and is associated with progression and poor prognosis in many types of cancer. Because of many similarities between highly dedifferentiated cancer cells and normal pluripotent stem cells, we applied human induced pluripotent stem cells (hiPSC) as a model for stemness studies. For the first time in hiPSC, we analyzed the function of individual TRIM28 domains. Here we demonstrate the essential role of a really interesting new gene (RING) domain and plant homeodomain (PHD) in regulating pluripotency maintenance and self-renewal capacity of hiPSC. Our data indicate that mutation within the RING or PHD domain leads to the loss of stem cell phenotypes and downregulation of the FGF signaling. Moreover, impairment of RING or PHD domain results in decreased proliferation and impedes embryoid body formation. In opposition to previous data indicating the impact of phosphorylation on TRIM28 function, our data suggest that TRIM28 phosphorylation does not significantly affect the pluripotency and self-renewal maintenance of hiPSC. Of note, iPSC with disrupted RING and PHD functions display downregulation of genes associated with tumor metastasis, which are considered important targets in cancer treatment. Our data suggest the potential use of RING and PHD domains of TRIM28 as targets in cancer therapy.

Sign in / Sign up

Export Citation Format

Share Document