Optimization of Schwann Cell Differentiation from Porcine Stem Cells

Abstract

Swine as a translational animal model of cancer is becoming increasingly recognized for its value in closing the gap between basic research and the development of clinically effective therapies. While rodent models have helped elucidate the complexities of many mechanistic pathways of cancer, they often have physiological and genetic differences that greatly limit the translatability of results to humans. The recognition of the value of swine models has also created a need for swine‐based in vitro cell culture platforms for mechanistic studies and drug screening. Such platforms are particularly sought after for tackling Schwannomas (Schwann cell based tumors) that are associated with diseases like neurofibromatosis 1 (NF1), a genetic disease caused by a mutation of the neurofibromin 1 (nf1) gene. These tumors are often benign, however, a small portion of them become malignant, degenerating into a form of cancer called neurofibrosarcoma. Patients with NF1 often develop neurofibromas from Schwann cells that can become malignant peripheral nerve sheath tumors, a tumor type that carries poor prognosis. Schwann cells, like most cells of a neuronal lineage, are challenging to culture in vitro. This has slowed progress of understanding the mechanisms responsible for these types of cancers. We therefore aim to develop an in vitro culture platform using swine cells to overcome these obstacles. This necessitated the optimization of culturing conditions to differentiate porcine stem cells into Schwann cells and then develop tumorigenic stem cells by the manipulation of the nf1 gene.We isolated adipose‐derived mesenchymal stem cells and testis‐derived spermatogonial stem cells for use in our experiments. Culturing parameters manipulated include temperature, matrix composition, growth factor levels, feeder cell status, and passaging conditions. In the subsequent phase, different stem cell transfection methods were studied with the goal of delivering CRISPR/Cas9 components targeting the nf1 gene. Techniques compared include lipofection using Lipofectamine 2000 and 3000 and electroporation using the Gene Pulser II or Neon Transfection System. Our findings will help researchers establish a more robust in vitro model for culturing Schwann cells from swine stem cells to study cancer development.Support or Funding InformationThe research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF Network, NF Central Plains, NF Michigan, NF Northeast, and Washington State NF Family.