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 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.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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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