Single-injection ex ovo transplantation method for broad spinal cord engraftment of human pluripotent stem cell-derived motor neurons

Publication date: Available online 3 February 2018
Source:Journal of Neuroscience Methods
Author(s): Maria C. Estevez-Silva, Akshitha Sreeram, Stephanie Cuskey, Nikolai Fedorchak, Nisha Iyer, Randolph S. Ashton
BackgroundTransplantation 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 methodWe 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.ResultsA 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.Comparison with existing method(s)Our single injection hMN transplant method is the first to standardize the optimal hMN phenotype for chick embryo transplantation, provide a rubric for engraftment quantification, and enable broad engraftment throughout the spinal cord with a single surgical intervention.ConclusionTransplantation of HB9⁺/ChAT⁻ hMN precursors into chick embryos of Hamburger Hamilton (HH) stages 15-18 using a single luminal injection confers a high probability of embryo survival and cell engraftment in diverse regions throughout the spinal cord.



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