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 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 cells5, 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 modeling7,8,9. 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 detected12,13,14.
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.
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
2. Porcine iPSCs induction into NPCs
3. NPC passaging
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, 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.
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 reported4,18 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.
The authors have nothing to disclose.
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).
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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