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

Summary

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

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

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.......

Materials

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