Elsevier

Stem Cell Research

Volume 7, Issue 2, September 2011, Pages 124-136
Stem Cell Research

Regular Article
Directed differentiation of porcine epiblast-derived neural progenitor cells into neurons and glia

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Abstract

Neural progenitor cells (NPCs) are promising candidates for cell-based therapy of neurodegenerative diseases; however, safety concerns must be addressed through transplantation studies in large animal models, such as the pig. The aim of this study was to derive NPCs from porcine blastocysts and evaluate their in-vitro differentiation potential. Epiblasts were manually isolated from expanded hatched blastocysts and cultured on MEF feeder cells. Outgrowth colonies were passaged to MS5 cells and rosettes were further passaged to Matrigel-coated dishes containing bFGF and EGF. Three NPC lines were established which showed expression of SOX2, NESTIN and VIMENTIN. One line was characterised in more detail, retaining a normal karyotype and proliferating for more than three months in culture. Following differentiation, TUJI was significantly up-regulated in protocol 2 (RA and SHH; 58% positive cells) as were NF and TH. In contrast, MBP was significantly up-regulated in protocol 3 (FGF8 and SHH; 63% positive cells), whereas, GFAP was significantly up-regulated in protocols 1–4 (33%, 25%, 43% and 22%). The present study provides the first report of a porcine blastocyst-derived NPC line capable of differentiating into both neurons and glia, which may be of paramount importance for future transplantation studies in large animal models of neurodegenerative diseases.

Highlights

►Three porcine neural progenitor cell (NPC) lines were derived from epiblast cells. ► They all express the markers SOX2, NESTIN, VIMENTIN and KI67. ► One detailed analyzed NPC line could be differentiated into neural and glial subtypes. ► The NPCs may be used in future transplantation experiments in large animal models.

Abbreviations

AA
Ascorbic acid
BDNF
Brain-derived neurotrophic factor
bFGF
Basic fibroblast growth factor
ESC
Embryonic stem cell
FGF8
Fibroblast growth factor 8
GDNF
Glial cell line-derived neurotrophic factor
ICM
Inner cell mass
iPSC
Induced pluripotent stem cell
LIF
Leukemia inhibitory factor
MEF
Mouse embryonic fibroblast
MPTP
1 - methyl 4 - phenyl - 1, 2, 3, 6 - tetrahydropyridine
MS5
Mouse stromal cell line 5
MZ
Marginal zone
NPC
Neural progenitor cell
OC
Outgrowth colony
PDGF
Platelet-derived growth factor
RA
Retinoic Acid
SHH
Sonic Hedgehog
SGZ
Subgranular zone
SZ
Subventricular zone
VZ
Ventricular zone

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