(10d) Immobilized NRG1-Fc Enhances Differentiation of Human Epidermal Neural Crest to Schwann Cells and Promotes Radial Sorting | AIChE

(10d) Immobilized NRG1-Fc Enhances Differentiation of Human Epidermal Neural Crest to Schwann Cells and Promotes Radial Sorting


Tseropoulos, G. - Presenter, University at Buffalo
Andreadis, S., State Univ of New York-Buffalo
Schwann Cell (SC) differentiation from Neural Crest (NC) Stem Cells plays a pivotal role in the myelination of the peripheral nervous system during embryonic development. However to date, the differentiation towards mature and functional human Schwann cells, for the purposes of cell transplantation or myelination drug testing assay development, has been elusive. Recently, we identified an easily accessible source of multipotent SOX10+/PAX3+/FOXD3+ NC cells isolated from human inter-follicular Keratinocytes (KC) isolated from glabrous neonatal foreskin. Transcriptomic analysis utilizing single cell RNA-seq, showed upregulation of pre-SC genes in specific k-means generated cell clusters indicative of the propensity of these cells to differentiate into myelinating SC (Fig. 1a). In order to recapitulate the in vivo SC differentiation signal topology, radial sorting and myelination, fusion NRG1-Fc proteins were immobilized on a hydrophobic polystyrene surface. Immobilized NRG1 on the surface induces sustained phosphorylation of the pathways downstream of the ERBb2/3, namely ERK1/2, MAP38 and AKT (Fig. 1b), which, in turn, lead to the upregulation of mature SC markers. Indeed after two weeks of culture on NRG1-Fc surface, mature SC markers PLP1, PMP22, KROX20 and MPZ were significantly upregulated with respect to differentiation on collagen coated surface (Fig. 1c). q-RT-PCR verify the transcriptional upregulation of SC mature markers and downregulation of dedifferentiation genes (c-Jun, CDK1 and FGF2) (Fig. 1d). In order to show functionality of our cells, we attempted to imitate the in vivo process of radial sorting, where SC choose which neuronal axon to myelinate. For this reason, we used a fibrous surface coated with a variety of extracelluar and signaling molecules. Indeed, the mRNA levels of SC markers, indicative of the cytoskeletal reformation (α7β1, Proffilin, N-WASp, CDC42), are upregulated and the differentiated cells show better alignment on the fibers than undifferentiated NC (Fig. 1e). Finally, SC differentiated on NRG1-Fc were plated on rat DRG neurons and showed decreased proliferation and increased alignment with respect to control (Fig. 1f).

Taken together our data shows that differentiation on NRG1-Fc substrate leads to faster SC maturation due to sustained phosphorylation of ERBb2/3 pathway. Continuous differentiation on fibers increases SC specific cytoskeletal markers leading to more efficient radial sorting. Our current work focuses on the in vitro myelination of PLA fibers or rat DRG neurons as well as transplantation of SC in the corpus callosum of the myelin deficient shiverer (shi/shi) mouse model. Given the accessibility of human skin, our SC differentiated from epidermal NC represent a potentially useful source of autologous multipotent stem cells for treatment of demyelinating diseases or spinal cord injuries, as well as potential in vitro demyelination assays that can be used for drug development.