Epigenetic Reprogramming Without Genetic Modification: Use of Sendai Virus Vectors for Generating Safe Induced Pluripotent Stem Cells

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.

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Sanfilippo syndrome type B: Reprogramming cultured skin fibroblasts to induced pluripotent stem cells using non-integrating Sendai virus vector

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2014.12.271 ◽

2015 ◽

Vol 114 (2) ◽

pp. S118

Author(s):

Kelly A. Turner ◽

Francis Y. Choy

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Sendai Virus ◽

Virus Vector ◽

Syndrome Type ◽

Type B ◽

Sanfilippo Syndrome ◽

Cultured Skin ◽

Induced Pluripotent

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Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Journal of Visualized Experiments ◽

10.3791/51406 ◽

2014 ◽

Cited By ~ 4

Author(s):

In Young Choi ◽

HoTae Lim ◽

Gabsang Lee

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Sendai Virus ◽

Somatic Cells ◽

Efficient Generation ◽

Induced Pluripotent

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

10.1101/2020.08.12.247619 ◽

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.

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Integration-Free Reprogramming of Human Somatic Cells to Induced Pluripotent Stem Cells (iPSCs) Without Viral Vectors, Recombinant DNA, and Genetic Modification

Methods in Molecular Biology - Engineering and Analyzing Multicellular Systems ◽

10.1007/978-1-4939-0554-6_6 ◽

2014 ◽

pp. 75-94 ◽

Cited By ~ 9

Author(s):

Boon Chin Heng ◽

Martin Fussenegger

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Genetic Modification ◽

Pluripotent Stem Cells ◽

Viral Vectors ◽

Recombinant Dna ◽

Somatic Cells ◽

Induced Pluripotent

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Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.806785 ◽

2022 ◽

Vol 8 ◽

Author(s):

Warunya Chakritbudsabong ◽

Ladawan Sariya ◽

Phakhin Jantahiran ◽

Nattarun Chaisilp ◽

Somjit Chaiwattanarungruengpaisan ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Sendai Virus ◽

Genetic Alterations ◽

Human Medicine ◽

Large Animal ◽

Induced Pluripotent ◽

Induced Neural Stem Cells

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.

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