University of Wisconsin–Madison

Randy Ashton, PhD – Slide of the Week

Ashton Slide of the Week

Title: Determining the optimal stage of motor neuron differentiation for ex ovo transplantation

Legend: (A) Rubric for hMN engraftment quantification. Scale 0-4 levels of CMPTX+/GFP+ hPSC-derived motor neuron (hMN) engraftment 48 hrs post-transplantation: no cells present (Scale 0); cells only in central canal (Scale 1); cells migrated into neural tube parenchyma (Scale 2, arrows); cells project GFP+ axons (Scale 3, arrows); cells project GFP+ axons into the ventral root (Scale 4, arrows). Scale bars are 100mm. Quantification of average percentage of tissue sections with Scale 1-4 engraftment (n=10 embryos per experimental group). * indicates p≤0.05 using a Wilcoxon Rank Sum Test with a post-hoc Benjamini-Hochberg procedure. (B) Immunostaining of hMN progenitor (Day 6), precursor (Day 16), and mature neuronal cultures (Day 30). Scale bars are 150 (i, ii, iii, iv) and 100mm (v, vi, vii, viii, ix).

Citation: Estevez-Silva MC, Sreeram A, Cuskey S, Fedorchak N, Iyer N, Ashton RS (2018). Single-injection ex ovo transplantation method for broad spinal cord engraftment of human pluripotent stem cell-derived motor neurons. J Neurosci Meth 298: 16-23.

Abstract: Background: Transplantation of human pluripotent stem cell (hPSC)-derived neurons into chick embryos is an established preliminary assay to evaluate engraftment potential. Yet, with recent advances in deriving diverse human neuronal subtypes, optimizing and standardizing such transplantation methodology for specific subtypes at their correlated anatomical sites is still required. New Method: We determined the optimal stage of hPSC-derived motor neuron (hMN) differentiation for ex ovo transplantation, and developed a single injection protocol that implants hMNs throughout the spinal cord enabling broad regional engraftment possibilities. Results: A single injection into the neural tube lumen yielded a 100% chick embryo survival and successful transplantation rate with MN engraftment observed from the rostral cervical through caudal lumbar spinal cord. Transplantation of HB9+/ChAT hMN precursors yielded the greatest amount of engraftment compared to Pax6+/Nkx6.1+/Olig2+ progenitors or mature HB9+/ChAT+ hMNs.

About the Lab: Our goal is to understand, model, and recapitulate in vitro the instructive signals utilized by human embryos to pattern tissue-specific differentiation of pluripotent stem cells, and apply this knowledge towards the rational design of tissue engineered scaffolds and other regenerative therapeutic strategies. Currently, we primarily focus on generating tissues and therapies for the central nervous system. For more information visit the Ashton Lab website.

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