606. cGMP Compliant Process to Produce Day-14 Transplantable Dopaminergic Neuron Precursors

Abstract

One of the key issues to bringing human embryonic stem cell (hESC)-based technology from bench to bedside is developing defined hESC culture processes and differentiation processes to produce sufficient amount of therapeutically relevant cells under Current Good Manufacture Practice (cGMP). Here we report a successful adaptation of a laboratory procedure into a scalable and cGMP compliant process, focusing on optimization of the process for day-14 transplantable dopaminergic (DA) neuron precursor production and the selection of cGMP-compliant reagents. We showed the equivalency between laboratory procedure and the cGMP process generated DA progenitors. The cells generated under cGMP compliant process exhibited similar culture growth characteristic and marker expression as the cells produced by the laboratory procedure as demonstrated by the following analysis: FACS analysis of PAX6 expression as a marker for neural stem cell (NSC); immunocytochemistry staining of TH and B-tubulin-III as marker for dopaminergic neuron; RT-qPCR of DBH and SERT as contaminant markers of noradrenergic and hindbrain neurons. In addition, we demonstrated the feasibility of generating a large intermediate cell bank (ICB) of neural stem cell for the production of DA neuron precursors. A cryopreserved ICB would greatly streamline the manufacturing process and reduce the lot-to-lot variability. In conclusion, we have successfully optimized and adapted a laboratory procedure for producing day-14 transplantable DA neuron precursor into scalable cGMP-compliant manufacture process which would provide great potential for clinical application.