HUMAN ES AND IPS CELL DIFFERENTIATION TO NEURAL PHENOTYPES.

We have developed a simple method to generate multipotent neural stem cells (termed EZ spheres) from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) without utilizing embryoid body formation. After gently lifting hESC or hiPSC colonies and culturing in suspension medium containing high concentrations of EGF and bFGF, cell aggregates, termed EZ spheres, form within 1 week with a propensity towards the neural lineage. EZ spheres allow simple and rapid proliferation of multipotent NSC populations from iPSC colonies in the absence of feeder layers, expensive substrates or complex serum components. EZ spheres show chromosomal stability over at least 30-40 passages, unlike some of the neural progenitor cell lines that we have extensively expanded for clinical grade cell banks. EZ spheres can subsequently be differentiated into neural and glial fates including dopamine, motor and striatal neurons, oligodendrocytes, and astrocytes. Using a transgene integration-free method of reprogramming normal episomal iPSC lines have been generated from human fetal neural progenitors (hNPC) with high efficiency. EZ spheres derived from hNPC-iPSCs have a significantly greater yield of neural cells upon differentiation. Using episomal pig iPSC-derived EZ sphere lines, we are establishing and validating the autologous engraftment of iPSC-derived motor neurons. EZ sphere cultures do not require complex combinations of substrates or morphogens, and need only twice weekly feedings. This allows for rapid growth of neural cells for developmental studies, high-content drug screening, or regenerative therapies. In addition, we are establishing episomal pig iPSC-derived EZ sphere lines to validate autologous engraftment of iPSC-derived motor neurons in large animals for cellular regenerative therapies in motor neuron disorders. We are now also exploring the innovative new world of direct reprogramming technology to efficiently reprogram across lineages to produce neural cells. [ABSTRACT FROM AUTHOR]