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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Differentiation of Porcine Induced Pluripotent Stem Cells (piPSCs) into Neural Progenitor Cells (NPCs)

Published: June 11th, 2021

DOI:

10.3791/62209

1Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, 2Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, 3Department of Pharmacology, Institute of Biosciences, São Paulo State University

This protocol describes a method for chemical differentiation and culture of neural progenitor cells derived from porcine induced pluripotent stem cells (piPSCs).

iPSC-derived neurons are attractive in vitro models to study neurogenesis and early phenotypic changes in mental illness, mainly when most animal models used in pre-clinical research, such as rodents, are not able to meet the criteria to translate the findings to the clinic. Non-human primates, canines, and porcine are considered more adequate models for biomedical research and drug development purposes, mainly due to their physiological, genetic, and anatomical similarities to humans. The swine model has gained particular interest in translational neuroscience, enabling safety and allotransplantation testing. Herein the generation of porcine iPSCs is described along with its further differentiation into neural progenitor cells (NPCs). The generated cells expressed NPC markers Nestin and GFAP, confirmed by RT-qPCR, and were positive for Nestin, b-Tubulin III, and Vimentin by immunofluorescence. These results show the evidence for the generation of NPC-like cells after in vitro induction with chemical inhibitors from a large animal model, an interesting and adequate model for regenerative and translational medicine research.

Even though many researchers aim to better understand the cellular mechanisms and pathological development of neurological diseases on humans, there are many limitations to using non-invasive techniques on humans such as magnetic resonance imaging (MRI), and the impossibility, in most cases, of applying invasive techniques such as tract-tracing and intracellular recording1. It is also challenging to obtain post-mortem brain tissue of good quality since prolonged agonal states of donors may affect the brain and interfere with the studies2. Therefore, there is a necessity for animal models, which have been used for several....

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These experiments were approved by the Ethics Committee on Animal Experimentation of the Faculty of Animal Science and Food Engineering, University of São Paulo (permit numbers: n° 5130110517 and n°4134290716).

NOTE: All procedures involving cellular culture and incubations are performed in a controlled atmosphere (38.5 °C and 20% CO2 in air, maximum humidity). Cellular passaging was performed by 5 min incubation with dissociation reagent and cells were recovered.......

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Characterization of piPSC
The characterization aimed to determine the acquisition of pluripotency of the reprogrammed cells. For that purpose, embryoid formation, immunofluorescence staining for pluripotency markers, and gene expression and spontaneous differentiation into embryoid bodies (EBs) were performed.

Generated cell colonies presented a flat, compact morphology in cell clusters with well-defined borders, as expected for piPSCs16,

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Through this protocol, fibroblasts were in vitro reprogrammed using the exogenous expression of OCT4, SOX2, c-MYC, and KLF4. The reprogrammed cells were maintained in vitro for more than 20 passages. When these lineages were submitted to the neuronal differentiation using chemical inhibitors, they expressed the neuronal progenitor cells' markers Nestin and GFAP, confirmed by RT-qPCR, and were positive for Nestin, β-Tubulin III, and Vimentin by immunofluorescence. Interestingly.......

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Prof. Kristine Freude is acknowledged for the assistance with protocols and scientific discussions. This work was financially supported by grants from the São Paulo Research Foundation (FAPESP) (# 2015/26818-5, # 2017/13973-8 and # 2017/02159-8), the National Council for Scientific and Technological Development (CNPq # 433133/2018-0), and the Coordination for the Improvement of Higher Education Personnel (CAPES) (financing code 001).

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Name Company Catalog Number Comments
293FT Invitrogen # R70007
6 well plates Costar # 3516
anti-B-Tubulin III abcam # ab7751
anti-NANOG abcam # ab77095
anti-NESTIN Millipore # ABD69
anti-OCT4 Santa Cruz biotechnology # SC8628
anti-SOX2 abcam # ab97959
anti-SSEA1 Millipore # MAB4301
anti-TRA1-60 Millipore # MAB4360
anti-VIMENTIN abcam # ab8069
B27-Minus Vitamin A Life Technologies # 12587010
DMEM/F-12 Life Technologies # 11960
donkey anti-goat 488 Invitrogen #  A11055
EGF Sigma # E9644
Fetal Bovine Serum Gibco # 10099
FGF2 Peprotech # 100-18B
GlutaMAX Gibco # 35050-061
Glutamine Gibco # 25030-081
goat anti-mouse 594 Invitrogen #  A21044
goat anti-rabbit 488 Invitrogen # A11008
Hexadimethrine bromide Sigma Aldrich # 107689
HighCapacity  kit Life Technologies # 4368814
IMDM Gibco # 12200-036
KnockOut DMEM/F-12 Gibco # 12660-012
Knockout serum replacement Gibco # 10828-028
LDN-193189 Sigma-Aldrich # SML0559
Leukocyte Alkaline Phosphatase kit Sigma Aldrich # 86R
Lipofectamine P3000™ Invitrogen # L3000-015
Matrigel Corning # 354277
Mitomycin C Sigma Aldrich # M4287-2MG
N2 Life Technologies # 17502048
Nanodrop ND-1000 Nanodrop Technologies, Inc.
Neurobasal medium Life Technologies # 21103049
Non-essential amino-acids Gibco # 11140-050
Penicillin-Streptomycin Gibco # 15140-122
Revita Cell Gibco # A2644501
Rnase out Life Technologies # 10777019
SB431542 Stemgent # 72232
StemPro Accutase Gibco # A11105-01
SW28 rotor Beckman Coulter # 342207
Trizol Life Technologies # 15596026
TrypLE Express Gibco # 12604-021
β-mercaptoethanol Gibco # 21985-023

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