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Stem Cells Transl Med. 2017 Mar;6(3):937-948. doi: 10.5966/sctm.2016-0073. Epub 2016 Oct 14.

Efficiently Specified Ventral Midbrain Dopamine Neurons from Human Pluripotent Stem Cells Under Xeno-Free Conditions Restore Motor Deficits in Parkinsonian Rodents.

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The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia.
Murdoch Children's Research Institute, The Royal Children's Hospital, Melbourne, Victoria, Australia.
Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia.
Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia.
Monash Institute of Pharmaceutical Sciences, Monash University, Clayton, Victoria, Australia.


Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)-derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson's disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock-in lines (LMX1A-eGFP and PITX3-eGFP) for in-depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this "next generation" protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements 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 demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC-derived neurons into the clinic. Stem Cells Translational Medicine 2017;6:937-948.


Dopamine neurons; Human pluripotent stem cells; LMX1A; PITX3; Parkinson’s disease; Ventral midbrain; Xenogeneic-free

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