ES cell-derived renewable and functional midbraindopaminergic progenitorsSangmi Chunga,b,1, Jung-Il Moona,b, Amanda Leunga,b, Daniel Aldricha,b, Stefan Lukianova,b, Yui Kitayamaa,b,Sara Parka,b, Yan Lic, Vadim Y. Bolshakovc, Thomas Lamoneried, and Kwang-Soo Kima,b,1

aMolecular Neurobiology Laboratory and Department of Psychiatry and Program in Neuroscience, bHarvard Stem Cell Institute, and cCellular NeurobiologyLaboratory and Department of Psychiatry, McLean Hospital/Harvard Medical School, Belmont, MA 02478; and dInstitute of Developmental Biology andCancer, Unité Mixte de Recherche, Centre National de la Recherche Scientifique 6543, Université de Nice–Sophia Antipolis, 06108 Nice, France

Edited by Fred H. Gage, The Salk Institute, San Diego, CA, and approved May 4, 2011 (received for review November 3, 2010)

During early development, midbrain dopaminergic (mDA) neuro-nal progenitors (NPs) arise from the ventral mesencephalic area bythe combined actions of secreted factors and their downstreamtranscription factors. These mDA NPs proliferate, migrate to theirfinal destinations, and develop into mature mDA neurons in thesubstantia nigra and the ventral tegmental area. Here, we showthat such authentic mDA NPs can be efficiently isolated fromdifferentiated ES cells (ESCs) using a FACS method combining twomarkers, Otx2 and Corin. Purified Otx2+Corin+ cells coexpressedother mDA NP markers, including FoxA2, Lmx1b, and Glast. Usingoptimized culture conditions, these mDA NPs continuously pro-liferated up to 4 wk with almost 1,000-fold expansion withoutsignificant changes in their phenotype. Furthermore, upon differ-entiation, Otx2+Corin+ cells efficiently generated mDA neurons,as evidenced by coexpression of mDA neuronal markers (e.g.,TH, Pitx3, Nurr1, and Lmx1b) and physiological functions (e.g.,efficient DA secretion and uptake). Notably, these mDA NPs dif-ferentiated into a relatively homogenous DA population with fewserotonergic neurons. When transplanted into PD model animals,aphakia mice, and 6-OHDA–lesioned rats, mDA NPs differentiatedinto mDA neurons in vivo and generated well-integrated DAgrafts, resulting in significant improvement in motor dysfunctionswithout tumor formation. Furthermore, grafted Otx2+Corin+ cellsexhibited significant migratory function in the host striatum,reaching >3.3 mm length in the entire striatum. We propose thatfunctional and expandable mDA NPs can be efficiently isolated bythis unique strategy and will serve as useful tools in regenerativemedicine, bioassay, and drug screening.

neural precursors | dopaminergic neurons | transplantation

Dysfunction of mDA neurons has been implicated in variousbrain diseases, such as neurodegenerative and psychiatric

disorders, for which there are no effective treatments. Thus,characterization and purification of mDA NPs will be crucial foran in-depth understanding of development, cellular properties,and survival of mDA neurons, and may translate into noveltherapeutic approaches for these diseases. During early braindevelopment, mDA neurons originate from the ventral midlineof the mesencephalon. The initial event of mDA neuron de-velopment depends on signaling molecules [e.g., Sonic hedgehog(Shh), fibroblast growth factor 8 (FGF8), and Wnt1] releasedfrom neighboring organizers, such as Notochord and Isthmus,setting up the initial field for mDA progenitors (1). These ex-trinsic signals initiate the regulatory cascades leading to mDAdevelopment by inducing key transcription factors, includingFoxA1/A2, Otx2, and Lmx1a/1b in mDA NP domains, followedlater by Nurr1 and Pitx3 in mDA neuronal domains (1, 2). Amongthese key transcription factors, FoxA2 plays an important rolein inducing floor plate cells (3), which were recently shown tobe mDA progenitors (4, 5). Another transcription factor, Otx2, isspecifically expressed in the NP domain of forebrain andmidbrainand can induce the mesencephalic floor plate phenotype whenectopically expressed in the more caudal floor plate (4).Based on these developmental studies, we hypothesize that

there are transient but authentic mDA progenitors with theability to proliferate, to differentiate into mDA neurons, and to

migrate to their final locations. To address this, we sought topurify mDA NPs by focusing on the coexpression of Corin,a floor plate cell-surface marker, and Otx2, a transcription factorthat can anteriorize floor plate cells to become mDA NPs. Be-cause they are expressed in other tissues, neither marker is suf-ficient to purify mDA NPs. Whereas Corin is also expressed inheart and skin (6), Otx2 is also expressed in forebrain and dorsalneural tube (7). To test our hypothesis that mDA NPs can beidentified by coexpression of Corin and Otx2, we performed themDA NP purification strategy and isolated Corin+Otx2+ cells.Here, we show that the purified mDA NP cells can be expandedand cryopreserved without losing their proliferation and differ-entiation potential; efficiently differentiate into authentic andfunctional mDA neurons; generate nondisruptive DA-enrichedgrafts upon transplantation; and significantly improve behavioraldysfunction in rodent PD models.

ResultsmDA NPs Can Be Identified by Coexpression of Otx2 and Corin bothin Vivo and in Vitro. During early mouse CNS development, Otx2is expressed in forebrain and midbrain (7), whereas Corin isexpressed in floor plate (4). Based on these observations (Fig.1A), we hypothesize that authentic mDA NPs are identifiable bycoexpression of these two markers. In support of this, mDA NPsin the ventral mesencephalon coexpress Otx2 and Corin at E10.5of mouse embryonic brain (Fig. 1 B–D). To test whether thesemDA NPs can be selected from ESCs, we used an embryoid body(EB)-based five-stage in vitro differentiation procedure (8) (Fig.S1A), which is known to well recapitulate early embryonic de-velopment. To specifically mark Otx2-expressing cells, we usedOtx2GFP knock-in (KI) ESCs (9). Although Otx2 expression isnot readily detectable at the ESC stage, it was robustly expressedfollowing in vitro differentiation at stage 4 (NP stage), faithfullyaccompanied by Otx2GFP expression (Fig. 1 E and F). Corin isundetectable in ESCs, but as cells differentiated, some expres-sion was observed (Fig. 1 G and H). At this stage, Otx2+Corin+cells were detected only as a minor population (Fig. 1H), becausethese markers are also expressed in alternate fate cells, such asOtx2+ forebrain cells (Pax6+) or dorsal neural tubes (Pax7+),and Corin+ caudal cord floor plate cells (Caro2+) or cardiaccells (SMA+; Fig. S1 B–E), thus justifying our strategy to useboth markers for specific purification of these mDA NPs.Following in vitro differentiation of Otx2GFP KI ESCs, we

observed high proportions of Otx2GFP+ cells, but very few ofthem were Corin+. Thus, to optimize ventralization, differenti-ating ESCs were treated with Shh conditioned media either atstage 3 (when NP cells start to emerge from EBs) or at stage 4

Author contributions: S.C., J.-I.M., Y.L., V.Y.B., and K.-S.K. designed research; S.C., J.-I.M.,A.L., D.A., S.L., Y.K., S.P., and Y.L. performed research; T.L. contributed new reagents/analytic tools; S.C., J.-I.M., A.L., D.A., S.L., Y.K., S.P., Y.L., V.Y.B., and K.-S.K. analyzed data;and S.C. and K.-S.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: schung@mclean.harvard.edu orkskim@mclean.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1016443108/-/DCSupplemental.

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(when NP cells are well established; Fig. 1I). Expression of bothFoxA2 and Corin was significantly increased only when the cellswere treated with conditioned medium at stage 3 (Fig. 1I andFig. S1 F–J), suggesting that there is a time window when dif-ferentiating ESCs are responsive to Shh-mediated ventralization.After this optimized in vitro differentiation, Otx2GFP KI

ESC-derived NP cells were subjected to FACS sorting followingstaining with anti-Corin antibody (Fig. S2). As shown in Fig. 2 A–Pand Fig. S3, Otx2+Corin+ cells were dramatically enriched bythis double FACS sorting. Furthermore, double FACS sortingalmost completely removed SSEA1+ pluripotent cells, which cancause teratoma formation after transplantation [Fig. 2 Q and R;14.7 ± 2.1% SSEA1+ in unsorted vs. <0.1% (detection limit)SSEA1+ cells in sorted Otx2+Corin+ cells]. Notably, the vastmajority of these sorted Otx2+Corin+ cells coexpressed otherknown mDANPmarkers, such as FoxA2 and Lmx1b (Fig. 2 S–V),confirming their identity as mDA NPs. They also express the NPmarker (nestin; 95.9 ± 1.3% Nestin+/total cells) as well as theradial glial marker (Glast; 89.9 ± 2.2% Glast+/total cells), butare largely negative for neuronal (β-tubulin; 3.5 ± 0.8% β-tubulin+/total cells) and glial markers (GFAP; 0.7 ± 0.5% GFAP+/totalcells; Fig. 2 W–BB), indicating that these cells represent immatureprogenitor cells.

Otx2+Corin+ Cells Efficiently Generate mDA Neurons but FewSerotonergic Neurons. Next, we differentiated sorted Otx2+Corin+ cells by mitogen removal and culturing in ND media for7 d [Fig. S1A; neuronal differentiation (ND) stage]. Initially, wefound that Otx2+Corin+ cells are very susceptible and prone tomassive cell death during neuronal differentiation following mi-togen removal (see below). Because this phenomenon was notobserved during neuronal differentiation of unsorted cells, wehypothesized that there may be cell survival factor(s) releasedfrom the unsorted mixed population. To test this possibility, wetreated differentiating Otx2+Corin+ cells with conditioned mediafrom differentiating unsortedmixed cells (ND-conditionedmedia).Indeed, treatment of Otx2+Corin+ cells with ND-conditionedmedia prominently increased their survival during differentiation(Fig. S4 A–C). We also found that cell-plating density is a criticalfactor for survival of purified cells during differentiation (Fig. S4D–F). Thus, we used higher plating density for differentiation ofpurified cells (>2 × 105 cells/cm2). Using these optimized differ-entiation conditions, we found that Otx2+Corin+ cells efficientlygenerated TH+ cells, whereas single-positive or double-negativecells were inefficient at generating TH+ cells (Fig. 3 A–D).Importantly, the vast majority of TH+ cells from Otx2+Corin+

cells coexpressed the mDA neuronal marker Pitx3 (Fig. 3 E–G;

83 ± 6.5% Pitx3+/TH+ cells), supporting the use of Otx2/Corindouble positivity to prospectively identify authentic mDA NPs.In addition, these TH+ neurons coexpressed additional mDAneuronal markers, Lmx1b and Nurr1 (Fig. 3 H and I; 88.9 ±1.5% Lmx1b+/TH+ cells). Furthermore, coexpression of thedopamine active transporter (DAT) and dopa decarboxylase(DDC; Fig. 3J) is evidence of their mature DA neuronal phe-notype. Purified Otx2+Corin+ cells generated both A9-like (44.5 ±5.1% Aldh1a1+/TH+ cells) and A10-like (56.3 ± 9.5% Calbin-din+/TH+ cells) mDA neurons (Fig. 3 K and L). Compared withunsorted cells, Otx2+Corin+ cells generated a significantly higherproportion of DA neurons, whereas the proportion of GABAneurons was significantly lower (Fig. 3 M–P and U; 5.6 ± 1.9% vs.80.2 ± 4.9% TH+/β-tubulin+ and 36.9 ± 4.9% vs. 8.7 ± 0.8%GABA+/β-tubulin+ for unsorted vs. sorted cells). Few DA neu-rons overlapped with GABA, suggesting these DA neurons are notof olfactory or forebrain origin (Fig. 3 Q and R). Remarkably, inthe purified cell population, serotonergic neurons were barelydetected (Fig. 3 S–U; 5.5 ± 40.7% vs. 0.01 ± 0.01% 5HT+/β-tubulin+ for unsorted vs. sorted cells).When analyzed for the ability to release DA in response to

membrane depolarization, Otx2+Corin+ cells released a signifi-cantly higher amount of DA compared with single-positive cells ordouble-negative cells (Fig. 3V). In addition, DA uptake by dif-ferentiated cells was much more prominent in differentiatedOtx2+Corin+ cells than single-positive or double-negative cells(Fig. 3W). To further test the functionality of Otx2+Corin+ cells,we performed whole-cell patch-clamp recordings. The recordedcells exhibited action potentials (spikes) upon injections of de-polarizing currents (Fig. 3 X and Y). The action potentials had anaverage amplitude of 72.0± 4.4 mV and a duration of 5.7± 0.9 ms(n = 7 cells); they were followed by the large afterhyper-polarization (AHP; amplitude: 9.4 ± 3.1 mV; duration: 54.9 ± 9.4ms, n = 7 cells). We have also observed the spontaneous excit-atory postsynaptic currents (sEPSCs) in recorded neurons duringcontinuous recordings at a holding potential of –70 mV (Fig. 3Z–AA). The sEPSCs exhibited fast kinetics (the rising time of the

Fig. 1. mDA NPs are identifiable by coexpression of Otx2 and Corin both invivo and in vitro. (A) Schematic diagram of Otx2 and Corin expression duringembryonic development. (B–D) Otx2 and Corin expression on E10.5 mousemDA NP domain. (E–H) Expression of Otx2, Otx2GFP, and Corin during ESCdifferentiation. (Scale bar: 50 μm.) (I) Induction of floor plate phenotype byShh-conditioned media treatment at stage 3 or stage 4 of differentiation,assayed by real-time PCR (mean ± SEM; n = 4).

Fig. 2. Purification of mDA NPs using coexpression of Otx2GFP and Corin.(A–P) Immunocytochemistry on FACS-purified cells using anti-Corin antibodyand anti-Otx2 antibody. (Q and R) FACS purification of mDA NPs efficientlyremoved residual pluripotent (SSEA1+) cells. (S–V) Purified Otx2+Corin+

cells express other mDA NP markers such as FoxA2 and Lmx1b. (W–BB)Purified Otx2+Corin+ cells also express Nestin and Glast, whereas there areonly few cells that express mature neural markers β-tubulin and GFAP. (Scalebar: 50 μm.)

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sEPCSs was 0.67± 0.11ms, and the decay time was 1.84± 0.26ms,n = 5 cells). These findings indicate that Otx2+Corin+-derivedneurons developed functional membrane properties. Taken to-gether, our data strongly suggest that Otx2+Corin+ cells repre-sent authentic mDA NPs that can generate a high proportion ofmDA neurons while dramatically diminishing other subtypeneurons, such as GABA and serotonergic neurons.

Self-Renewability andDifferentiation Potential of Purified Otx2+Corin+

Cells Can Be Supported by bFGF and FGF8.We next investigated theself-renewability (or expandability) of Otx2+Corin+ cells in re-sponse to various signaling molecules. We tested those factorsimplicated in the regulation of mDA NPs (e.g., Shh, FGF8, Wnt1,and Wnt5a) (2, 10–14) or the proliferation of general NPs (e.g.,bFGF, EGF, Dll4, and Jag1) (15–18) as well as FGF20, whichwas implicated in mDA survival (19). Each factor was added fora week to mitogen-free media (ND media), which by itself does

not support the proliferation of purified cells. At a concentrationof 50 ng/mL, only bFGF (FGF2) and FGF8 supported pro-liferation of Otx2+Corin+ cells, but not Shh, FGF20, Wnt1,Wnt5a, EGF, Dll4, or Jag1 (Fig. 4A). Only bFGF and FGF8generated a large proportion of Ki67+-proliferating cells (Fig. 4B–K and Fig. S5A). In contrast, differentiated TH+ cells weregreatly increased in the presence of other factors (Fig. 4 L–U andFig. S5B). These findings suggest that bFGF and FGF8 supportthe self-renewal of mDA NP, while preventing them from dif-ferentiating. In support of this notion, after 1 wk, bFGF- orFGF8-treated cells showed the presence of an enriched Nestin+cell population, whereas cells treated with the other factorsshowed enriched β-tubulin+ neurons compared with Nestin+cells (Fig. S5 C–L).Because bFGF most efficiently supported proliferation of

Otx2+Corin+ cells, we tested their extended proliferation in thepresence of bFGF. We found an ∼1,000-fold increase after 4 wk(Fig. 5A). Remarkably, these expanded cells largely coexpressedthe mDA NP markers Otx2, FoxA2, and Corin (Fig. 5 B–E andFig. S6 A and B; 80.1 ± 5.8% Otx2+ cells and 80.4 ± 1.7%FoxA2+ cells), as well as Glast and Nestin (Fig. 5 F–I and Fig.S6B). Neural differentiation of expanded cells also generated ahigh proportion of DA neurons (Fig. 5 J–O and Fig. S6C; 75.6 ±2.9% TH+/β-tubulin+ cells) without notable changes in otherneural cell types (Fig. 5 P–T and Fig. S6C). Further immuno-cytochemical analysis showed that expanded cells generatedmDA neurons, as evidenced by coexpression of mDA markers(e.g., Pitx3, Nurr1, and Lmx1b; Fig. 5 U–X and CC–FF and Fig.S6C) and coexpression of mature DA markers (e.g., DAT andDDC; Fig. 5 Y–BB). Expanded cells also generated both A9-likeand A10-like mDA neurons (Fig. 5 GG–HH).We next tested whether the addition of other factors with

bFGF can further enhance the proliferation of Otx2+Corin+cells. We found that the addition of FGF8 further enhancedtheir proliferation, suggesting that FGF8 and bFGF work in-dependently through different receptors (20) (Fig. S7A). Inter-estingly, Shh, EGF, Dll4, and Jag1, which are unable to supportthe proliferation of purified cells alone, further enhanced theirproliferation (Fig. S7A) without significant differences in theproportion of apoptotic cells (Fig. S7 D–M). None of thesefactors significantly changed the phenotype of Otx2+Corin+cells, as examined by the proportion of FoxA2+ and Otx2+ cells(Fig. S7 B and C).Wealso testedwhetherOtx2+Corin+ cells can be cryopreserved

without losing their properties. FACS-purified Otx2+Corin+ cells

Fig. 3. Purified Otx2+Corin+ cells generate mDA neurons. (A–D) TH immu-nocytochemistry. (E–J) Purified Otx2+Corin+ cells generated mature mDAneurons that coexpress Pitx3, Lmx1b, Nurr1, DAT, and DDC. (K and L) Puri-fied cells generated DA neurons with both A9-like (Aldh1a1+) and A10-like(Calbindin+) phenotype. (M–R) FACS purification significantly enrich DAneurons, and significantly decrease GABAergic neurons. (S and T) Seroto-nergic neurons were efficiently removed after double FACS sorting. (Scalebar: 50 μm.) (U) Cell-counting analysis before and after FACS (mean ± SEM;n = 4). Unsorted or Otx2+Corin+ cells at ND stage were analyzed for DAdifferentiation, GABAergic differentiation, or serotonergic differentiation.(V) DA release analysis (mean ± SEM; n = 4). (W) Specific DA uptake by DAT(mean ± SEM; n = 4). (X) The injection of depolarizing current (10 pA) topurified cells at the ND stage led to the firing of action potentials by therecorded cell under current-clamp conditions. (Y) The first spike from X ona faster time scale. The action potential is followed by the large AHP. (Z)Representative traces showing sEPSCs under control conditions. (AA) Tracesshow sEPSCs recorded from a single cell, which were aligned by their onset.

Fig. 4. bFGF and FGF8 support proliferation of purified mDA NPs. (A) Foldincrease of purified mDA NPs in the presence of various signaling molecules(mean ± SEM; n = 4). (B–K) Cells treated with bFGF or FGF8 show robustproliferation as shown by Ki67 staining. (L–U) Immunocytochemistry analysisshowing that in the absence of an mDA NP proliferating signal, bFGF, orFGF8, purified cells spontaneously differentiated into DA neurons, as shownby TH staining. (Scale bar: 50 μm.)

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before and after expansion were cryopreserved in 90% FBS and10% DMSO and recovered at 3, 6, and 12 mo. Thawed cells werecultured in NP media and differentiated in vitro using the sameprocedure (Fig. S1A). Remarkably, the cells exhibited similarproliferation and differentiation properties as the originalOtx2+Corin+ cells, largely coexpressing Otx2 and FoxA2 at theNP stage (78.7± 5.3%Otx2+/total cells and 80.2± 2.3% FoxA2+/total cells) and efficiently generating DA neurons (86.2 ± 0.9%TH+/b-tubulin+ cells) upon mitogen removal (Fig. S8). Thesedata suggest that purified Otx2+Corin+ cells maintain their cel-lular and differentiation properties upon long-term storage.

In Vivo Transplantation of Otx2+Corin+ Cells. We tested the func-tionality of Otx2+Corin+ cells in vivo following transplantationinto mouse striatum. To optimize transplantation conditions, wefirst transplanted purified cells either at the NP stage or at day 3of the ND stage into the striatum of normal mice. Grafts wereanalyzed 7 wk after transplantation. The number of surviving DAneurons was significantly lower with ND-stage cells comparedwith NP-stage cells (Fig. 6A). We next attempted to transplantsorted cells to the striatum of Pitx3-deficient aphakia (ak) micethat were previously shown to have a major loss of A9 mDAneurons, leading to motor deficits that are reversed by L-DOPA(21). We transplanted NP-stage Otx2+Corin+ cells into thestriatum of ak mice, followed by a series of basal ganglia-relatedbehavioral analyses at 4 and 6 wk following transplantation (21).

When placed head upward on top of a vertical pole, ak micetransplanted with Otx2+Corin+ cells took much less time toorient themselves downward than control ak mice (Fig. 6B).Total latency to travel downward also showed consistent resultsas head-down measurements (Fig. 6C). In addition, ak micetransplanted with Otx2+Corin+ cells required significantly lesstravel time on the challenging beam compared with the mock-transplanted group (Fig. 6D). These results strongly suggest thattransplanted ak mice perform significantly better than their age-matched control ak mice on a battery of behavioral tests that aresensitive to defects of the nigrostriatal DA system.Seven weeks after transplantation, transplanted mice were

killed and analyzed by immunohistochemistry. Whereas micetransplanted with Otx−Corin− cells often showed disruptivegrafts largely devoid of DA neurons (Fig. 6 E and F and Fig.S9A), mice transplanted with double-positive cells showed well-integrated grafts with enriched DA neurons (Fig. 6 G and H).Consistent with this, stereological analysis showed that the graftsize of double-negative cells is 6.9 ± 3 mm3, whereas that ofdouble-positive cells is 0.10 ± 0.012 mm3 (Fig. 6I). However,total DA neurons were significantly higher in double-positivecells compared with double-negative cells (Fig. 6J; 545.3 ± 73.6DA neurons vs. 2.3 ± 1.0 neurons in double-positive grafts vs.double-negative grafts) with DA density of 5,738.4 ± 459.8/mm3

vs. 7.2 ± 3.5/mm3 in double-positive grafts vs. double-negativegrafts (Fig. 6K). Interestingly, Otx2+Corin+ cells exhibitedprominent migratory function within adult striatum, as evidencedby the presence of DA neurons across the entire striatal sectionsspanning more than 3.3 mm (330× the size of the neuronal cellbody) even outside the graft core (Fig. 6 L–AA). Fig. 6BB showsa mock-transplanted brain without any DA neuronal cell body.When transplanted with GFP-labeled Otx2+Corin+ cells, weobserved migrated cells expressing both GFP and TH, showingthe graft-derived origin of migrated DA neurons (Fig. S9 B–G).We further tested in vivo function of Otx2+Corin+ cells after

transplantation into the striatum of 6-OHDA–lesioned rats.Compared with control rats undergoing sham surgery, trans-planted rats showed significant motor improvement in bothamphetamine-induced rotation and use of lesioned paw by cyl-inder test (Fig. 6 CC–DD). Histological analysis showed that theygenerated well-integrated graft with enriched DA neurons (Fig.S9 Q–T) with 1,509.0 ± 401.11 TH+ cells/graft and graft size of0.369 ± 0.136 mm3. Grafted cells extended TH fiber outside thegraft core into the host (Fig. S9U and Fig. 6EE) and formedsynaptic connections, as indicated by synaptophysin+ synapticstructure next to TH+ cell body (Fig. 6FF). Transplanted NPcells matured in vivo to express both TH and DDC (Fig. 6 HH–JJ), with both A9 (Girk2+) and A10 (Calbindin+) DA neurons(Fig. 6 KK–PP; 53.6 ± 5.8% Girk2+/TH+ cells and 49.1 ± 3.3%Calbindin+/TH+ cells). The majority of these DA neurons in thegraft coexpressed the mDA neuronal markers Lmx1b, Pitx3,En1, and FoxA2 (Fig. 6 QQ–TT in rats; 87.3 ± 2.2% Lmx1b+/TH+ cells, 83.0 ± 1.7% Pitx3+/TH+ cells, and 93.2 ± 1.7%FoxA2+/TH+ cells; Fig. S9 H–P in mice), showing they differ-entiated into mature mDA neurons in vivo.

DiscussionTo realize the potentials of pluripotent stem cells such as ESCsand induced pluripotent stem cells (iPSCs) in regenerativemedicine, it is crucial to establish efficient differentiation meth-ods into appropriate cell lineages. Toward this goal, a straight-forward approach is to direct in vitro differentiation to a specificcell lineage using physiological signaling molecules and/or keytranscription factors. This strategy has been successfully used forthe efficient generation of several neuronal cells, such as dopa-mine and motor neurons (22–27). However, this strategy haslimitations such as nonexpandability, high vulnerability, and/orheterogeneity of the differentiated neuronal cells. An alternateapproach consists of identifying and generating specific NP cells.This approach has recently been used to generate Purkinje cellprogenitors or floor plate cells (28, 29). Previously, we purifiedSox1+ NPs from differentiating ES cells, but these cell pop-ulations inefficiently generated DA neurons (30), possibly due tothe nature of mDA NPs as glia-like floor plate cells rather than

Fig. 5. Purified mDA NPs can be expanded in vitro without losing theirdevelopmental potential. (A) Growth curve of purified mDA NPs in thepresence of bFGF (mean ± SEM; n = 3). (B–I) The majority of mDA NPs expressFoxA2, Otx2, Nestin, and Glast after 4 wk of expansion. (J–O) Purified mDANPs can generate DA neurons after 4 wk of expansion in bFGF coexpressingTH and β-tubulin. (P–T) The proportion of neural subtypes does not changesignificantly over expansion (mean ± SEM; n = 4). (U–HH) Further charac-terization of ND cells derived from 4 wk-expanded mDA NPs coexpressing THwith Pitx3, DAT, DDC, Nurr1, and Lmx1b. Expanded cells also generate bothA9 and A10 phenotype. (Scale bar: 50 μm.)

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Sox1+ NPs (4). These findings led us to hypothesize that it iscritical to use developmentally appropriate marker(s) to identifyand purify functional and authentic subtype-specific NPs.To address these critical issues, we focused on mDA neurons

and their hypothetical progenitors. In particular, we hypothesizedthat prospective mDA NPs are identifiable by the coexpressionpattern of two specific markers, the floor plate marker Corin andthe midbrain/forebrain-specific transcription factor Otx2. Usingthis double-selection strategy, we successfully identified func-tional and expandable mDA NPs. First, we found that purifiedOtx2+Corin+ cells coexpress other genes that are known to bespecifically expressed in mDANPs, including FoxA2, Lmx1b, andGlast. Second, following in vitro neuronal differentiation, thesecells efficiently generated mDA neurons that coexpress midbrainneuronal markers (e.g., Pitx3, Nurr1, DAT, and DDC) and ex-hibited physiological functions (e.g., DA secretion, DA uptake,and action potential). Finally, upon transplantation into thestriatum of two rodent PD models, Otx2+Corin+ cells differen-tiated into mDA neurons in vivo and significantly improvedmotordysfunction with well-integrated grafts and remarkable migrationcapacity in the host striatum. Taken together, we conclude thatESC-derived Otx2+Corin+ cells represent authentic mDA NPswith proper molecular, cellular, and differentiation properties.Our results show that these purified cells are very useful to

investigate the biological properties of mDA NPs. For instance,we found that Otx2+Corin+ mDA NPs can proliferate withoutspontaneous differentiation if bFGF or FGF8 was added tominimal ND medium. In contrast, no such effect was observed

when Shh, FGF20, Wnt1, Wnt5a, EGF, Dll4, or Jag1 wereadded, although these factors were previously associated with NPproliferative activities. Notably, previous studies of mDA NPproliferation mostly relied on animal system(s) or mixed primarycultures, sometimes resulting in conflicting observations. Forexample, even though bFGF is widely used as a mitogen for NPs(16), mDA NPs increased in bFGF KO mice, whereas mDA NPsdecreased in bFGF-overexpressing transgenic mice (15, 31).These unexpected results may be caused by multiple compen-satory mechanisms in vivo. In our purified mDA NPs study, weclearly observed that bFGF could remarkably support pro-liferation of mDA NP on its own. We also noted that FGF8could support proliferation of mDA NPs, which is in agreementwith previous studies (13). Interestingly, FGF20, which is im-plicated in mDA survival (19), did not support proliferation ofmDA NPs, showing specificity among FGF family members insupporting mDA NP proliferation.Another salient finding of this study is that purified mDA NPs

are highly self-renewable while maintaining their differentiationpotential, which will be critical for cell replacement therapy ofPD. In sharp contrast to nonexpandable mDA neurons, ourresults demonstrate that purified mDA NPs are expandable forat least up to 4 wk with almost 1,000-fold expansion, and couldbe frozen for long-term storage and thawed without changes intheir phenotypes and developmental potential. These findingsstrongly support that these mDA NPs represent a potential cellsource. In support of this, our transplantation study showed thatOtx2+Corin+ mDANPs not only generated well-integrated grafts

Fig. 6. Purified mDA NPs generate functional neurons in vivofollowing transplantation. (A) Otx2+Corin+ cells transplantedat NP vs. ND stage (mean ± SEM; n = 7). (B–D) mDA NPs, whentransplanted into ak mice, can ameliorate the behavioraldeficit. Rd1 mice were used as a blind mice control (mean ±SEM; n = 10). (E–H) Otx2−Corin− cells generated large graftslargely devoid of DA neurons, whereas Otx2+Corin+ cellsgenerated well-integrated graft, enriched in DA neurons, asshown by TH staining. Dotted line marks graft. Inset is shownmagnified on Right. Otx2−Corin− graft in the wild-type host ispresented to show the disruption of host striosome structureand absence of DA neurons in the graft by TH staining.Otx2+Corin+ graft in the ak host is shown to better visualizethe integration of DA graft with few host TH fiber back-ground. (I) Graft volume (mean ± SEM; n = 10). (J) Total TH+

cells in the graft (mean ± SEM; n = 10). (K) DA density withingraft (mean ± SEM; n = 10). (L–AA) Otx2+Corin+ mDA NPsshow migratory ability, shown by migration of DA neuronsacross almost the entire striatum (shown is a slide from everysixth coronal section). (BB) Mock-transplanted striatum. (CC–DD) mDA NP, when transplanted into 6-OHDA–lesioned rats,can significantly improve behavioral deficits, as shown byamphetamine-induced rotational behavior and cylinder test(mean ± SEM; n = 6). (EE–FF) Transplanted mDA NPs integrateinto host striatum, as shown by TH fiber innervation andsynapse formation. (GG–II) Transplanted mDA NPs generatemature mDA neurons in vivo coexpressing TH and DDC. (JJ–OO) Transplanted mDA NPs generated both A9 and A10 DAneurons. (PP–SS) Transplanted mDA NPs generated mDAneurons as shown by coexpression of TH with Lmx1b, Pitx3,En1, and FoxA2. (Scale bars: red, 500 μm; black or white, 50 μm.)

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with migrating mDA neurons but also avoided tumor formationin host animals, which is in sharp contrast to transplantation ofCorin−Otx2− or nonsorted NPs. In addition, transplantation ofmDA NPs resulted in much better survival compared withtransplantation of differentiated mDA neurons. Furthermore, wefound that mDA NPs differentiate into a remarkably homoge-neous population. In particular, our double-sorting scheme al-most completely removed serotonergic neurons, which have beenshown to cause graft-induced dyskinesia (32, 33). Because theconditions that induce mDA neurons from ES cells also induceserotonergic neurons (34), our purification scheme is an impor-tant step in clinical application of pluripotent stem cells for PD.During development, serotonergic neurons are generated nextto mDA domains in the ventral basal plate of rostral hindbrainright outside the floor plate. Thus, it appears that our Otx2 andCorin double-selection effectively removed serotonergic pro-genitors by removing both hindbrain and nonfloor plate cells.In conclusion, we have shown that the double-selection strat-

egy using Corin and Otx2 efficiently purifies functional and ex-pandable mDA NPs that may serve as useful tools to furtherinvestigate mDA NPs biology, which will translate into effectivecell replacement therapy for PD and other degenerative diseases.Toward these goals, it will be crucial to test if the same double-selection strategy can also work with human pluripotent stemcells to purify human mDA NPs. Recently, an efficient way togenerate the KI reporter in human ESCs was developed (35) thatcan help generate the Otx2GFP reporter in the human system.Preferably, the identification of a cell surface marker combina-tion that can prospectively identify mDA NPs would facilitatethe purification of hESC- or hiPSC-derived mDA NPs for bio-medical and clinical applications.

MethodsESC Culture and in Vitro Differentiation. ES cell cultures were maintained anddifferentiated as described previously (9). Detailed information can be foundin SI Methods.

FACS Purification. Differentiated Otx2GFP ESCs were subject to FACS sortingfollowed by anti-Corin staining at the end of stage 3. Detailed informationcan be found in SI Methods.

Real-Time PCR Analysis, Immunocytochemistry, and Immunohistochemistry.Real-time PCR analysis, immunocytochemistry, and immunohistochemistryanalysis was done as described previously (2). Detailed information can befound in SI Methods.

DA Release Assay, DA Reuptake Assay, and Electrophysiology Analysis. The DArelease assay was performed as described previously (8), and detailed in-formation can be found in SI Methods. For DA reuptake assay, double-positive–, single-positive–, or double-negative–derived ND cells (ND stageday 7) were used for DA reuptake assay, and detailed information can befound in SI Methods. For electrophysiology analysis, FACS-sorted cells weredifferentiated for 2 wk and then subject to electrophysiology analysis. De-tailed information can be found in SI Methods.

Transplantation Analysis and Behavioral Analysis. FACS-purified Otx2+Corin+

cells, expanded for 4 d in NP media, were used for transplantation, followedby behavioral analysis, as described in detail in SI Methods.

ACKNOWLEDGMENTS. This study was supported by National Institutes ofHealth Grants NS070577, MH087903, and MH048866.

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