Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerativemedicine.Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind.Whereasmouse PSCderived DA neurons have shown efficacy in models of Parkinson’s disease,DAneurons fromhumanPSCsgenerally showpoor invivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of humanDA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSCderived midbrain DA neurons. In vivo survival and function is demonstrated inParkinson’s diseasemodels using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimbuse and akinesia. Finally, scalability is demonstrated by transplantation intoparkinsonianmonkeys. ExcellentDAneuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson’s disease. Recent mouse genetic studies have demonstrated an important role for the transcription factor FOXA2 during midbrain DA neuron development. A unique feature of the developing midbrain is the coexpression of the floor-plate (FP) marker FOXA2 and the roof plate marker LMX1A. Normally, FP and roof plate cells are located at distinct positions in the central nervous system (ventral versus dorsal) and show diametrically opposed patterning requirements. We recently reported the derivation of FP precursors from ES cells using a modified dual-SMAD inhibition protocol. Canonical Wnt signalling is important for both roof plate function and midbrain DA neuron development. We therefore proposed that WNT activation may induce LMX1A expression and neurogenic conversion of PSCderived midbrain FP towards DA neuron fate. Here we report that exposure to CHIR99021 (CHIR), a potent GSK3B inhibitor known to strongly activate WNT signalling, induces LMX1A in FOXA21 FP precursors (Fig. 1a). CHIR was much more potent than recombinant WNT3A or WNT1 at inducing LMX1A expression (data not shown). The efficiency of LMX1A induction was dependent on the timing of CHIR exposure with a maximum effect from day 3 to day 11 (Supplementary Fig. 1). CHIR was the most critical factor for inducing coexpression of FOXA2/LMX1A, while other factors such as FGF8 had only marginal effects (Supplementary Fig. 2). Induction of FOXA2/ LMX1A coexpression required strong activation of SHH signalling using purmorphamine, a small molecule agonist, alone or in combination with recombinant SHH (Supplementary Fig. 3). Treatment with SHH agonists (purmorphamine1 SHH) and FGF8 (S/F8) in the absence of CHIR showed significantly lower expression of FOXA2 by day 11 and complete lack of LMX1A expression (Fig. 1a, b). Dual SMAD inhibition (exposure to LDN1931891 SB431542, ‘LSB’) did not yield FOXA2-expressing cells, but a subset of LMX1A1 cells (Fig. 1a, b). The anterior marker OTX2 was robustly induced in LSBand LSB/S/F8/CHIR-treated cultures, but not under LSB/S/F8 conditions (Fig. 1a, c). Systematic comparisons of the three culture conditions (Fig. 1d) were performed using global temporal gene expression profiling. Hierarchical clustering of differentially expressed genes segregated the three treatment conditions by day 11 of differentiation (Supplementary Fig. 4a). FOXA1, FOXA2 and several other SHH downstream targets including PTCH1 were among the most differentially regulated transcripts in LSB/S/F8/CHIR versus LSB treatment sets (Fig. 1e). Expression of LMX1A, NGN2 (also known as NEUROG2) andDDC indicated establishment ofmidbrainDAneuron precursor fate already by day 11 (Fig. 1e, f). In contrast, LSB cultures were enriched for dorsal forebrain precursor markers such as HES5, PAX6, LHX2 and EMX2. Direct comparison of LSB/S/F8/CHIR versus LSB/S/F8 treatment (Fig. 1f) confirmed selective enrichment for midbrain DA precursor markers in LSB/S/F8/CHIR group and suggested hypothalamic precursor identity in LSB/S/F8-treated cultures based on the differential expression of RAX, SIX3 and SIX6 (ref. 17) (see also POMC and OTP expression in Fig. 2d below). The full list of differentially expressed transcripts (Supplementary Tables 1 and 2) and gene ontology analysis (Supplementary Fig. 4b) (DAVID; http:// david.abcc.ncifcrf.gov, ref. 18) confirmed enrichment for canonical WNT signalling upon CHIR treatment (raw data available at the Gene Expression Omnibus). Comparative temporal gene expression analysis for markers of midbrain DA precursors (Fig. 1g) versus anterior and ventral non-DA fates (Fig. 1h) partitioned the three induction conditions into (1) LSB: dorsal forebrain; (2) LSB/S/F8: ventral/hypothalamic; (3) LSB/S/F8/CHIR: midbrain DA identity.