Neurons From Human Pluripotent Stem Cells Under Xeno-Free Conditions Restore Motor Deficits in Parkinsonian Rodents

In this study, the authors describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs. The protocol is translational across multiple embryonic and induced hPSC lines. hPSCs were cultured xeno-free on laminin-521 in TeSR2. For vmDA differentiation, two xeno-free matrix proteins, vitronectin, and human laminin-521 were compared for their ability to replace Matrigel. Both matrices facilitated appropriate patterning, however, only laminin-521 supported the long-term attachment of neural precursors. This “next-generation” protocol consistently increases both the yield and proportion of vmDAneural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. The mechanism underlying these improvements are identified and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno-free vmDA progenitors from LMX1A- and PITX3-eGFP reporter lines into Parkinsonian rodents demonstrate improved engraftment outcomes and restoration of motor deficits.