Efficient derivation of transgene-free porcine induced pluripotent stem cells enables in vitro modeling of species-specific developmental timing
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ABSTRACT:
PROVIDER: PRJNA947624 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23942300.fastq.gz | Fastqsanger.gz | |||
| SRR23942301.fastq.gz | Fastqsanger.gz | |||
| SRR23942302.fastq.gz | Fastqsanger.gz | |||
| SRR23942303.fastq.gz | Fastqsanger.gz | |||
| SRR23942304.fastq.gz | Fastqsanger.gz |
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Project description:Sus domesticus (pig) are an excellent large mammalian model for comparative studiesdue to their relatively comparable physiology and organ size to humans. The derivation of transgene-free porcine pluripotent stem cells (PiPSCs) will therefore benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. Established PiPSCs can robustly differentiate into derivates representing the primary germ layers in vitro and in vivo. Furthermore, the transgene- free PiPSCs preserve intrinsic species-specific developmental timing in culture. This capacity is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at ~ 3.7 hours, a time scale recapitulating in vivo porcine somitogenesis. We therefore propose that these transgene-free PiPSCs can serve as a powerful tool for modeling development and disease, informing both conserved and unique features of mammalian pluripotency and developmental timing mechanisms.
2023-11-09 | GSE227985 | GEO
Project description:Sus scrofa domesticus (pig) has served as a superb large mammalian model for biomedical studies because of its comparable physiology and organ size to humans. The derivation of transgene-free porcine induced pluripotent stem cells (PiPSCs) will, therefore, benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. PiPSCs can be differentiated into derivatives representing the primary germ layers in vitro and can form teratomas in immunocompromised mice. Furthermore, the transgene-free PiPSCs preserve intrinsic species-specific developmental timing in culture, known as developmental allochrony. This is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at ∼3.7 h, a timescale recapitulating in vivo porcine somitogenesis. We conclude that the transgene-free PiPSCs can serve as a powerful tool for modeling development and disease and developing transplantation strategies. We also anticipate that they will provide insights into conserved and unique features on the regulations of mammalian pluripotency and developmental timing mechanisms.
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Project description:Zebrafish has become a preeminent model for developmental biology research, largely due to the ease of transgenesis. Despite widespread usage of transgenic lines, mapping of transgene insertion sites is rare, which raises complications involving potential local chromatin influences on transgene expression, off-target effects, and issues with allelic variation. To address these short-comings, we introduce TransTag, a simple and efficient method utilizing Tn5 transposase-mediated tagmentation, for streamlined identification of Tol2-based transgene insertion sites in zebrafish. TransTag is straightforward to perform and can identify insertion sites without the need for alignment of raw sequencing data. We also provide a detailed protocol for TransTag, a step-by-step guide for data analysis, and a user-friendly Shiny app, making transgene mapping achievable at low cost for researchers without programming expertise. Altogether, TransTag emerges as a valuable tool to enhance the precision and utility of transgenesis studies by providing essential chromosome-specific information on transgene locations.
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Project description:We previously reported the non-viral derivation of transgene-free induced pluripotent stem cells (iPSCs) from somatic fibroblasts of a female beagle dog using an optimized induction medium and non-transgene-integrating episomal vectors. Here, we report derivation of an induced pluripotent stem cell line OF35Y-iPS from a male beagle dog, which showed standard characteristics of pluripotency such as a strong gene expression profile of pluripotency markers, differentiation potential into all three germ layers, and normal karyotype (78XY). Furthermore, we demonstrated the iPSC line was suitable for genome editing technology. The novel iPSC line would be a useful foothold for stem cell research and regenerative veterinary medicine
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Project description:Emergence of induced pluripotent stem cells (iPSC) technology has paved novel routes for regenerative medicine. iPSCs offer the possibilities of disease modeling, drug toxicity studies as well as cell replacement therapies by autologous transplantation. Classical protocols of iPSC generation harness infection by retro- or lenti-viruses. Although such integrating viruses represent very robust tools for reprogramming, the presence of viral transgenes in iPSCs is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Moreover, remaining reprogramming transgenes have been shown to affect the differentiation potential of iPSCs. More recently, alternative protocols have been explored to derive transgene-free iPSC, including use of transposons, mRNA transfection, episomal plasmid transfection, and infection with non-integrating viruses such as Sendai virus. However, the utility of such protocols remains limited due to low efficiency and narrow range of cell specificity. In this study we aim at combining the robustness of lentiviral reprogramming with the high efficacy of Cre recombinase protein transduction to readily delete reprogramming transgenes from iPSCs. We demonstrate rapid generation of transgene-free human iPSCs by excising the loxP-flanked reprogramming cassette employing direct delivery of biologically active Cre protein. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs do resemble more to human ESCs and has better differentiation potential than iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of superior transgene-free iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies. mRNA extracted from human Fibroblasts (AR1034ZIMA), human Embryonic Stem Cell line I3 (hES I3), three human induced Pluripotent Stem Cell clones 1, 1.2 and 1.4 (fl-ARiPS cl1, del-ARiPS cl 1.2, del-ARiPS cl1.4) has been hybridized on Illumina Human HT-12 (version 4 revision 2) arrays for genome wide expression analysis. Samples were run at least as duplicate technical replicates. Differential gene expression analysis has been performed on the grouped expression data with the human embryonic stem cells (hES I3) group as the reference.
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Project description:Emergence of induced pluripotent stem cells (iPSC) technology has paved novel routes for regenerative medicine. iPSCs offer the possibilities of disease modeling, drug toxicity studies as well as cell replacement therapies by autologous transplantation. Classical protocols of iPSC generation harness infection by retro- or lenti-viruses. Although such integrating viruses represent very robust tools for reprogramming, the presence of viral transgenes in iPSCs is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Moreover, remaining reprogramming transgenes have been shown to affect the differentiation potential of iPSCs. More recently, alternative protocols have been explored to derive transgene-free iPSC, including use of transposons, mRNA transfection, episomal plasmid transfection, and infection with non-integrating viruses such as Sendai virus. However, the utility of such protocols remains limited due to low efficiency and narrow range of cell specificity. In this study we aim at combining the robustness of lentiviral reprogramming with the high efficacy of Cre recombinase protein transduction to readily delete reprogramming transgenes from iPSCs. We demonstrate rapid generation of transgene-free human iPSCs by excising the loxP-flanked reprogramming cassette employing direct delivery of biologically active Cre protein. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs do resemble more to human ESCs and has better differentiation potential than iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of superior transgene-free iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies.
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