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

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.