Abstract
Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine-induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma-and Mad-related protein (SMAD) inhibitors, a TGF-β inhibitor and BMP4 inhibitor. The dSMADi-derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid-treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma-cultured medium including injury-related cytokines treated porcine iPSC-NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.
glioblastoma, induced pluripotent stem cells, neural progenitor cells, pig --

and Development Center (GRDC) Program through the NRF funded by the Ministry of Education in Korea, Award/Award Number: 2017K1A4A3014959
| INTRODUCTION
Directed neural differentiation of pluripotent stem cells (PSCs) enables researchers to generate diverse neuronal cell sources for neural development study and regenerative medicine. [1] [2] [3] While many studies have used rodent models of neurogenesis, these models are not suitable for preclinical studies in regenerative medicine that include neural stem cell therapies or for studying human neurodegenerative diseases, because they are poor representations of the human neural system. 4 There is therefore a need for the generation of an efficient in vitro platform for neural cell production in large animal models such as the most closely related non-primate animal species, which is the pig. 5 For example, pigs have similar genomic, anatomical, immunological and physiological characteristics to those of humans, as well as a comparable organ size and lifespan. [6] [7] [8] Furthermore, porcine models are cost-effective as they are by-products from the abattoir, thus they are widely available compared to other large animal models. In particular, there are similarities with humans in the growth pattern of the neonatal brain and extent of peak brain growth at the time of birth. 9, 10 Furthermore, pigs have gyrencephalic brain with similar grey and white matter composition and size that is more comparable to humans than are rodents. 11 Despite these advantages, there is lack of information about efficient direct neural differentiation in porcine model.
For the robust and reproducible neuronal differentiation of PSCs, the establishment of self-renewable neural progenitor cells (NPCs) is required. 12, 13 In general, most of PSCs can differentiate into NPCs, despite significant variability in methods and efficiency for neural induction systems. Although some strategies have been used to differentiate porcine pluripotent cells into neuronal populations, little is currently known about neural development in pigs. Puy et al reported the differentiation of porcine neural cells derived from the inner cell masses (ICMs) of blastocyst-stage embryos. 14 Other studies have reported the derivation of NPCs from porcine isolated epiblasts from blastocysts, and evaluated their in vitro differentiation potential into neuronal and glial cells. 15, 16 The majority of these initial reports have claimed that neural differentiation can be typically induced by the formation of the embryoid body (EB) 17 or that it uses undefined factors, such as animal feeder cell lines or components. 18 However, the EB formation method, which can form all three germ layers, produced only small numbers of neural commitment cells because of the mixed presence of other cell lineages, such as those of mesodermal or endodermal origin. Also, the inductive protocols for neuronal cells in such porcine research utilized a coculture system using a neural inducing 20 These are involved in critical tissue functions, including control of early development, and survival and differentiation of neurons and astrocytes. [21] [22] [23] Especially, GBM is a grade IV fast-growing glioma originated from the transformation of the normal primitive precursors of glial cells (glioblasts). 24 Based on this aspect, the potential impact of the factors derived from GBM on maturation and differentiation of progenitor cells needs to be uncovered.
The aim of this study was thus to examine the effect of endogenous and exogenous factors in the differentiation of porcine iPSCs into NPCs. It has been known that dual small-molecule targeting of SMAD signalling efficiently neuralizes human PSCs by blocking both BMP and Activin/Nodal pathways. 25 By combining dual SMAD inhibitors (dSMADi) such as LDN193189 or SB431542 with the control of seeding cell density, we generated high-yield NPCs from porcine iPSCs. The neuronal fate specification can be recapitulated in vitro using regional specific factors. In addition, the effects of brain cancer cell line-derived conditioned medium (CM) on the differentiation of iPSC-NPCs into neuronal-like cells were also examined to establish an efficient system for the generation of neural cells from porcine iPSCs.
| MATERIALS AND METHODS

| Ethics statement
This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Veterinary and Quarantine Service. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Chungbuk National University (permit number: CBNUA-584-13-01). All animal subjects were killed using isoflurane anaesthesia, and all efforts were made to minimize suffering. Experimental procedures for establishing GBM cell lines were approved by the Ethics Committee and permission was granted from the institutional review board KIM ET AL.
of Chungbuk National University Hospital (IRB No.: 2016-08-009-002). Written informed consent was obtained for all patient samples.
| Chemicals
Consumables were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise stated.
| Culture of porcine iPSCs
The porcine iPSCs used in this study were kindly provided by Pro- antibiotic-antimycotic (Gibco), 10 ng/mL EGF and 4 ng/mL bFGF (Invitrogen Corporation) at a 3% O 2 and 5% CO 2 atmosphere. After achieving 80%-90% confluency, the cultures were passaged at 1:3.
The medium was changed twice a week. To prepare GBM-CM, GBM cells were cultured on 100 mm culture dishes until confluent, and then the medium was replaced with 8 ml of fresh medium. After 24 hours of incubation at 37°C, the medium was collected, filtered and stored at −80°C for experiments.
| Alkaline phosphatase (AP) activity detection
Porcine iPSCs were harvested, washed three times with PBS and fixed in 4% paraformaldehyde for 5-7 minutes at room temperature.
AP activity was then assayed with NBT/BCIP chromagen solution (Roche, Basel, Switzerland).
| Karyotyping
To check for cytogenetic abnormalities, karyotyping of porcine iPSC or GBM cells were performed in accordance with standard cytogenetic techniques. 29 Briefly, confluent monolayers of porcine iPSCs were treated with 10 µg/mL colcemid (Gibco, Carlsbad, CA) for 3- with trypsin (Gibco) and stained with Giemsa (GTG-banding method).
Chromosomes were then counted under a standard bright field microscopy and checked for cytogenetic abnormalities.
| Gene expression analysis by real-time PCR or reverse transcription PCR
The Table S1 . The fluorescence intensity was measured at the end of the extension phase of each cycle with threshold values set manually. Relative expression was determined by the 2 Ct method, with RN18S as a control. Experiments were repeated at least three times.
| Immunofluorescence
Immunofluorescence (IF) was performed as follows: Cells were washed with 1x PBS containing Ca 2+ and Mg 2+ and fixed with 4%
paraformaldehyde. The cells were washed three times with PBS and permeabilized with 0.2% Triton X-100 for 5 minutes for intracellular markers analysis. The fixed cells were co-incubated with blocking solution (10% goat serum in PBS) and primary antibody overnight at 4°C. The primary antibodies used in this study are listed in Table S2 .
The following day, cells were washed three times with washing medium (Tween-20, Triton X-100 and PBS) and incubated with appropriate secondary antibodies at room temperature for 1 hour. Nuclei were then stained with Hoechst 33342 and the stained cells examined using a confocal microscope and ZEN 2009 Light Edition software (Carl Zeiss, Oberkochen, Germany). Figure S2A . After neural induction using dSMADi, the high-density group showed a significantly higher number of colonies than the low-density group (0.8 ± 0.4 vs 10.2 ± 0.9, P < 0.01, Figure S2B ).
| Statistical analysis

| Patterning and further differentiation of porcine iPSC-NPCs
To investigate whether porcine iPSC-NPCs are responsive to instructive regionalization cues, we exposed porcine iPSCs treated with Together, these data demonstrate that GBM cell-derived soluble factors may play a pivotal role in the extensive further differentiation of porcine iPSC-NPCs.
| DISCUSSION
In this study, we developed a novel platform for neural induction and differentiation of porcine NPCs and commitment progeny cells using dSMADi and patterning factors without EB formation or feeder cell coculture. Our system also included further differentiation from porcine iPSC-NPCs to neuronal cells using patient-derived GBM-CM.
Recent studies have indicated that the interactions between cells
and their microenvironment play critical roles in determining cell fate in vivo. 36, 37 Cells are affected by signalling factors such as soluble cytokines, cell-cell contacts and insoluble extracellular matrices. 38 Therefore, it was thought that specific neuronal fates are determined during nervous system development through co-ordinated At the same time, the initial cell density, which is one of the endogenous factors, has an important impact in the commitment of
PSCs into a particular cell lineage. 42 It is well known that the seeding cell density can result in variable terminal cell densities, giving rise to various outcome of differentiation. 25 In contrast to the MS5 protocol, which requires low plating density, the dSMADi method allowed for high plating densities. In the present study, the dSMAD inhibition group with low seeding density failed to initiate neural induction.
We found that the initial seeded cells with high density form-derived cells subsequently affected differentiation of the NE, the primordium of the nervous system. Therefore, the collaboration of dSMAD inhibition system and high cell density resulted in an efficient induction of early neural lineage from porcine iPSCs, which is consistent with a human study. 42 In terms of protein levels, the nuclear expression of the early NE marker, PAX6, was observed in the high cell density dSMADi group with the expression of CD133 and Vimentin.
Although the mRNA expression of placode marker, nerve growth factor receptor p75, was activated in the high cell seeding density group, there was no HNK1 protein, a marker for migrating NC cells at a detectable level. This indicates that neuroectodermal fate is enforced under the action of dSMAD inhibitors with high cell density under this protocol.
During embryonic developmental stages of body axis elongation, tissues express HOX genes along the rostrocaudal axis of the CNS. ; RA has been reported to be involved in anterior versus posterior patterning during CNS development. 45 Consistent with human studies, 46 current study showed that RA has a role in posterior neuronal specification during differentiation which indicates that the restricted regionalization of porcine iPSC-NPCs can be converted to caudal progenitors during patterning process using a defined morphogen. After exposure to ventralizing factors such as, SHH with RA, the expression of Tuj1 was increased. It is well known that SHH affects human neural cells in ventral patterning 47, 48 and generate midbrain/hindbrain dopaminergic neurons when combined with RA or FGF8 treatment. 49 Collectively, this study highlights the Although further studies are needed to definitively uncover this possibility, this is the first study to report the potential application of a differentiation-inducing effect of GBM-CM in porcine iPSC-NPCs.
The porcine iPSCs used in this study showed obvious expression in pluripotent markers such as POU5F1 and SSEA4, which are known to be characterized in other porcine iPSCs. [55] [56] [57] SSEA4+ porcine iPSCs are known to be highly capable of differentiating into neural cell types similar to in vivo-derived porcine NPCs. 58 However, one recent study has reported that the efficiency of deriving neural rosette (NR) structure, which consists of a radial arrangement of neuroepithelial cells, may vary for each cell line depending on the expression levels of POU5F1 and SSEA4. 59 According to this report, porcine iPSCs with low POU5F1 and high SEEA4 expression generated limited NR formation. However, we examined ZO-1 expression of NR structure in this study regardless of the relatively low expression of POU5F1.
In conclusion, we developed a robust and efficient platform to generate porcine NPCs from porcine iPSCs and commitment protocols that differentiate into neural and glial cells. The neural induction of porcine iPSCs into NPCs was based on the chemically defined 

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