Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus.

Warunya Chakritbudsabong,Nattarun Chaisilp,Ruttachuk Rungsiwiwut,Joao N Ferreira,Phakhin Jantahiran,Sasitorn Rungarunlert,Ladawan Sariya,Somjit Chaiwattanarungruengpaisan

doi:10.3389/fvets.2021.806785

Warunya Chakritbudsabong, Nattarun Chaisilp + Show 6 more

Open Access

https://doi.org/10.3389/fvets.2021.806785

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Abstract

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.

Highlights

The remarkable discovery that differentiated cells can be completely reprogrammed into induced pluripotent stem cells by viral-mediated transduction of exogenous transcription factors marks a significant breakthrough in regenerative medicine [1]. iPSCs offer an infinite supply of differentiated cells for various purposes, including disease modeling in vitro, drug research, toxicity testing, and autologous cell-based therapy [2]
The following day, the medium was switched to the induced neural stem cells (iNSCs) medium comprising DMEM/F-12 and a neurobasal medium at a ratio of 1:1 supplemented with 2% B-27 supplement, 1% N-2 supplement, 1% antibiotic-antimycotic solution, 1% GlutaMAX, 20 ng/mL human basic fibroblast growth factor, and 10 ng/mL human epidermal growth factor
To address the question of whether Porcine tail fibroblasts (PTFs) can be directly converted into stably expanding multipotent porcine iNSCs (piNSCs), PTFs

Summary

Introduction

The remarkable discovery that differentiated cells can be completely reprogrammed into induced pluripotent stem cells (iPSCs) by viral-mediated transduction of exogenous transcription factors marks a significant breakthrough in regenerative medicine [1]. iPSCs offer an infinite supply of differentiated cells for various purposes, including disease modeling in vitro, drug research, toxicity testing, and autologous cell-based therapy [2]. The forced expression of NSC transcription factors [9,10,11] or pluripotency transcription factors, encoding OCT4, SOX2, KLF4, and c-MYC (OSKM) [12,13,14], converts differentiated cells directly into induced neural stem cells (iNSCs) and induced neural progenitor cells (iNPCs). This approach is an appealing alternative to existing iPSC technology because it enables the production of patient-specific NSCs without passing through the pluripotent stage, thereby decreasing the tumorigenic risk [15, 16]. When iNSCs are transplanted into animal models for up to 6 months, they can alleviate disease phenotypes and prevent developing tumors, demonstrating their therapeutic promise for neurological disorders [23]

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