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

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 decades in translational research, being both relevant and questionable until today. The choice of a particular animal model is becoming a central question in recent experimental design and planning, making clear that in order to obtain consistent results, the selection of the most appropriate model requires profound knowledge not only of the physiology of the different species but also importantly, on the specific aims of the research³.

However, animal models frequently present limitations when capitulating the human brain structure and development since it has some unique developmental, anatomical, molecular, and genetic features. Therefore, it is somewhat difficult to interpret and extrapolate data gathered from animals used in research, such as data from rodents¹.

Among the wide variety of animal models available nowadays, including transgenic models, some large animals are considered highly valuable, such as non-human primates, canines, and porcine⁴. The physiological, genetic, and anatomical similarities between humans and porcine regarding organ size emphasize the significance of these models in developing diagnostic and therapeutic approaches. Especially, the swine model has gained particular interest in translational neuroscience, enabling safety and allotransplantation testing. It has been used in research related to cardiovascular, pulmonary, gastrointestinal affections, and, in particular, for testing new therapies (e.g., in regenerative medicine studies with stem cells^{5, 6}).

In this context, in vitro models, and more specifically induced pluripotent stem cells (iPSCs)-derived neurons, are attractive models for allowing the study of 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.

The use of iPSCs has greatly benefited neuroscience by allowing disease modeling in vitro, particularly by using iPSCs-derived neural progenitor cells (NPC), since NPCs have shown to be an interesting tool for in vitro disease modeling^7,8,9. iPSCs have been successfully generated from patients with Alzheimer's disease¹⁰, schizophrenia¹¹, and many other diseases such as Parkinson's disease, Rett syndrome, spinal muscular atrophy, Down syndrome, and amyotrophic lateral sclerosis as compiled by Mungenast and collaborators². Pre-clinical animal model systems have also been reported using iPSC-derived NPCs as functional spine grafts with minimal or no immune response detected^12,13,14.

Herein, the generation of porcine iPSCs and further chemical differentiation into putative neural progenitor cells is described (Figure 1 and Figure 2). The generated cells expressed NPC markers Nestin and GFAP, confirmed by RT-qPCR, and were positive for Nestin, β-Tubulin III, and Vimentin by immunofluorescence. These results show the evidence of the generation of NPC-like cells after in vitro induction with chemical inhibitors from a large animal model, an important and adequate model for regenerative and translational medicine research.

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 after centrifugation (300 x g).

1. Porcine fibroblast reprogramming into iPSC

1. Experiment preparation
   1. Prepare fibroblast and 293 culture media consisting of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum (FBS), 0.1 mM non-essential amino acids, and 1% antibiotics (penicillin/streptomycin) unless otherwise stated.
   2. Prepare iPSC culture medium (reprogramming medium) consisting of low osmolality DMEM/F12 (optimized for the growth of human embryonic and induced pluripotent stem cells) supplemented with 20% serum replacement, 0.1 mM non-essential amino acids, 1 mM glutamine, 3.85 µM β-mercaptoethanol, 10 ng/mL bFGF, and 1% antibiotics.
   3. When needed, seed mouse embryonic fibroblasts (MEFs) into a T75 flask (10 mL) to obtain 70-80% confluency in the following day (approximately 6 x 10⁵ per T75). The next day, inactivate by a 2 h 30 min incubation with 200 µL of 0.5 mg/mL mitomycin C (add mitomycin in the T75 containing MEFs without prior media change).
   4. After the incubation period, recover cells after incubation with a dissociation solution for 5 min and seed into wells previously coated with gelatin in a 6 well plate at a 1x10⁵ concentration.
   5. Coat wells by incubating it with an 0.1% gelatin solution for 20 min at 37 °C (approximately 1 mL of gelatin solution per well to cover the entire well) and immediately aspirate. Then, remove the solution and replace with culture medium (2 mL per well).
2. Transfection and lentiviral production
   1. Culture 293 cells in T75 culture flasks until it reaches approximately 90% confluency.
   2. Dissociate cells and seed 5 x 10⁶ cells per new T75 flask without antibiotics.
   3. The next day, prepare two solutions per flask (T75) for transfection: 1: 1.5 mL of IMDM (no antibiotics, no serum) with the appropriate concentration of each vector (12 µg of OSKM; 1.2 µg of TAT; 1.2 µg of REV; 1.2 µg of hgpm2, and 2.4 µg of VSVG_{2°generation,}); and 2: 1.5 mL of IMDM (no antibiotics, no serum) plus 36 µL of lipofection reagent (or as recommended by the manufacturer) ¹⁵.
   4. Mix the solutions and incubate for 15 min.
   5. Meanwhile, replace the medium of the 293 cells, adding only 7 mL of IMDM supplemented with 10% FBS per flask.
   6. After the incubation period, add 3 mL of the lipofection reagent + plasmids in each flask. Replace the medium with IMDM 10% FBS after 6 h (optional).
   7. Collect medium (complete volume - 10 mL per T75) at the 24, 48, and 72 h time points. Filter it with a 0.45 mm PVDF filter and weigh it for balancing before ultracentrifugation.
   8. Centrifuge for 1 h 30 min hour at 48,960 x g.
   9. Discard supernatant by pouring and incubate the remaining content (approximately 200 µL) for 1 h at 4 °C.
   10. Resuspend viral pellet delicately pipetting up and down several times and aliquot viral solution.
3. Transduction
   1. Seed 2x10⁴ fibroblasts per well of a 6-well plate. Include wells for molecular analysis (e.g., PCR) and transduction controls (e.g., GFP analyses) (optional).
   2. The following day, remove 1 mL of medium and add 1 µL of hexadimetrine bromide (8 µg/mL) and 50 µL of the viral solution.
   3. Incubate for 3 to 4 h, followed by a complete medium change (2 mL) (Day 0).
   4. Culture cells for 5 days, changing medium every 2 days.
   5. Previously prepare 0.1% gelatin-coated and MEF plates as described in item 1.1.3.
   6. To dissociate cells and replate cells in reprogramming medium, wash wells using 1 mL phosphate-buffered saline solution (PBS). Remove PBS.
   7. Add 1 mL of dissociation reagent per well and incubate the cells at 37 °C for 5 min.
   8. Transfer cells to a conical tube and centrifuge cells for 5 min at 300 x g and resuspend them in 1-3 mL of iPSC culture medium.
   9. Count the cells using a Neubauer chamber or a cell counter equipment and seed them at a 1-2 x 10⁴ concentration into previously 0.1% gelatin-coated and MEFs covered wells.
   10. Change iPSC culture medium every 2 days.
   11. iPSC colonies (at passage 0, P0) will appear at approximately 10 days of the reprogramming period. Manually pick morphologically typical colonies (round edges and cells with a high nuclear-cytoplasm ratio) using a 26 G needle to detach colony and surrounding MEFs.
   12. Transfer 1 colony per new well, individually, using a 10 or 100 µL pipette tip, for clonal culture and characterization of putative iPSC cell lines.
4. Porcine iPSC passaging
   1. Manual passaging and colony picking
      1. Clean and transfer an inverted microscope into a laminar flow hood.
      2. Sterilize the microscope with ultraviolet (UV) light for 15 min.
      3. Wash previously gelatin and MEF coated wells with PBS prior to colony transfer. Remove PBS.
      4. Add 2 mL of iPSC medium.
      5. Locate the colony of interest in the well that will be used.
      6. Using the bevel of an insulin syringe needle, separate the colony from surrounding cells.
      7. If the colony is small, detach it from the well by pipetting medium up and down its borders with a 10 µL pipette.
      8. If it is a larger colony, cut it into a few smaller segments with the needle.
      9. Aspirate the colony or colony's fragments with a 10 µL pipette.
      10. Transfer it to the new well.
   2. Enzymatic passaging of clonal lines
      1. Wash wells using PBS. Remove PBS.
      2. Add 1 mL of dissociation reagent per well and incubate the cells at 37 °C for 5 min.
      3. Transfer approximately 100 µL of cells to a new well. This amount may vary among different cell lines; therefore, daily visual analysis of confluency is highly recommended.

2. Porcine iPSCs induction into NPCs

1. Experiment preparation
   1. Prepare Neural Induction Medium (NIM) composed of 50% Neurobasal medium and 50% DMEM/F-12 supplemented with B27-Minus Vitamin A (20 µL/mL), N2 (10 µL/mL), 1 mM glutamine (10 µL/mL), penicillin-streptomycin (1 µL/mL). Filter the solution in a 0.22 µm filter and add BMP signaling inhibitor LDN193189 and ALK inhibitor SB431542 at a final concentration of 0.1 µM and 10 µM, respectively.
   2. Coat wells of a 6 wells plate with 1 mL of matrix solution and incubate at 37 °C for at least 30 minutes. Remove matrix solution and add E8 medium for iPSCs culture.
   3. On day 13 of the induction protocol, prepare Neural Expansion Medium (NEM) composed of 50% Neurobasal medium and 50% DMEM/F-12 supplemented with B27-Minus Vitamin A (20 µL/mL), N2 (10 µL/mL), NEAA (10 µL/mL), 1 mM glutamine (10 µL/mL), penicillin-streptomycin (1 µL/mL). Filter the solution in a 0.22 µm filter and add FGF2 and EGF to be at an end concentration of 10 ng/mL.
2. Induction protocol
   1. The day before the iPSCs reaches 100% confluency, transfer cells into a new well (1:1 split). Wash the well by adding 1 mL of PBS. Remove PBS.
   2. Add 1 mL of 0.5 mM EDTA and incubate cells at 37 °C for 5 min.
   3. Remove EDTA and add 1 mL of E8 culture medium.
   4. Gently wash cells off the well and transfer the contents into a new well previously coated with matrix solution.
   5. The following day, wash wells with PBS. Remove PBS and add 2 mL of NIM.
   6. Change induction medium every day for 14 days.
   7. On day 14, wash wells using PBS. Remove PBS.
   8. Add 1 mL of cell dissociation reagent per well and incubate the cells at 37 °C for 5 min.
   9. Transfer cells to a conical tube and centrifuge cells for 5 min at 300 x g and resuspend them in 6 mL of NEM medium.
   10. Add 60 µL (10 µL/mL) of a post-thaw recovery solution and transfer 2 mL of the solution to each matrix-coated well.
   11. Change NEM medium the next day, and then at every two days.
       ​NOTE: Each well that went through the induction protocol is considered to give rise to a new NPC line. Therefore, their respective cells should not be mixed.

3. NPC passaging

1. Wash well with 1 mL of PBS. Remove PBS.
2. Add 1 mL of cell dissociation reagent and incubate cells at 37 °C for 5 min.
3. Gently wash cells of the well and transfer the contents into a conical tube.
4. Centrifuge the solution for 5 min at 300 x g. Remove the supernatant and add 6 mL of NEM.
5. Gently homogenize the pellet and transfer 2 mL of the solution to a new well previously coated with matrix solution.

Representative Results

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 piPSCs^{16, 17} and very distinct from fibroblast morphology. They were alkaline phosphatase positive (Figure 3) and positive for OCT4, SOX2, and partially positive for SSEA1, and TRA 1-60 staining (Figure 4). These cells were able to form EBs and expressed endogenous pluripotency genes OCT4 and NANOG (Table 1).

Characterization of piPSC-derived neural progenitor cell
The characterization aimed to determine the neural lineage commitment of the generated cells. In this case, immunofluorescence staining for NPC markers as well as gene expression was performed.

Porcine iPSC-derived NPCs cells presented different morphology compared to the iPSC cells and were positively stained for Nestin, β-Tubulin III, and Vimentin, suggesting successful differentiation and neural lineage commitment (Figure 5). Also, the expression of transcripts for the NPC markers Nestin and GFAP was detected, which corroborates with the results obtained by immunostaining (Table 1).

Figure 1: Representative procedures of the processes of pig iPSC generation. Please click here to view a larger version of this figure.

Figure 2: Representative procedures of the processes of pig iPSC induction into neural differentiation. Please click here to view a larger version of this figure.

Figure 3: piPSCs and EBs. A: piPSCs in bright field (bar=200μm). B: Alkaline phosphatase detection (bar=400 μm). C: EBs (bar=400 μm). Please click here to view a larger version of this figure.

Figure 4: Representative image of piPSCs and immunostaining for OCT4+SOX2 (bar=100 μm), SSEA1 (bar=200 μm), and TRA-1-81 (bar=200 μm). Please click here to view a larger version of this figure.

Figure 5: A: Neuronal induction of piPSCs, NIM at D0 and D11, and NPCs-like at P2 (bar=200 μm). Immunofluorescence of NPCs markers: β Tubulin III (bar=100 μm), Vimentin (bar=200 μm) and Nestin (bar=100 μm). Please click here to view a larger version of this figure.

	Nestin	bIII-tubulin	Vimentin	GFAP
RT-qPCR	√	N/A	N/A	√
Immunostaining	√	√	√	N/A

Table 1: Summary of the main results after chemical differentiation of in vitro reprogrammed cells into NPCs-like.

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, possibly due to the embryonic nature of the reprogrammed porcine fibroblast used to differentiate into NPC-like cells, the presence and expression of Nestin were detected at different levels at all stages of reprogramming and differentiation. Indeed, the spatiotemporal expression of Nestin has been reported in neonatal and fetal fibroblasts in humans and swine.

Although several reports have already discussed the generation of putative piPSCs ^4,18, it is noteworthy that different pluripotency profiles may lead to potentially different differentiation outcomes. The lack of generation of bonafide piPSCs still hampers its full potential in regenerative medicine, although a great number of studies have already been reported^4,18 on the generation and use of these cells. An important question to be addressed is the limitation of the exogenous expression of pluripotency factors, often due to the use of integrative systems used to reprogram somatic cells in this species. The transgene expression may lead to a possible incomplete differentiation in vitro, and thus, functional and terminally differentiated neurons are still a drawback to be surpassed.

Herein we show that in the described conditions, putative NPCs were obtained in vitro using chemical induction. These results will contribute to using the porcine model in future regenerative and translational medicine research, paving the way to a future possible differentiation of in vitro reprogrammed cells into functional neuronal cells in this model.

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