Search

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. 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. Conclusion Transplantation 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.

The embryonic neuroepithelium gives rise to the entire central nervous system in vivo, making it an important tissue for developmental studies and a prospective cell source for regenerative applications. Current protocols for deriving homogenous neuroepithelial cultures from human pluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followed by a manual isolation step or adherent differentiation using small molecule inhibitors. Here, we report that hPSCs maintained under chemically defined, feeder-independent, and xeno-free conditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax6+/N-cadherin+ with widespread rosette formation) within 6 days under adherent conditions, without small molecule inhibitors, and using only minimalistic medium consisting of Dulbecco’s modified Eagle’s medium/F-12, sodium bicarbonate, selenium, ascorbic acid, transferrin, and insulin (i.e., E6 medium). Furthermore, we provide evidence that the defined culture conditions enable this high level of neural conversion in contrast to hPSCs maintained on mouse embryonic fibroblasts (MEFs). In addition, hPSCs previously maintained on MEFs could be rapidly converted to a neural compliant state upon transfer to these defined conditions while still maintaining their ability to generate all three germ layers. Overall, this fully defined and scalable protocol should be broadly useful for generating therapeutic neural cells for regenerative applications. Stem Cells 2014;32:1032–1042