Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1

Abstract

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson's disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1⁺ cells survive and differentiate into mDA neurons in vivo, resulting in a significant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1⁺ cells into the brain of an MPTP-treated monkey. Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.

ARTICLE

Received 14 Sep 2015 | Accepted 2 Sep 2016 | Published 14 Oct 2016

Bumpei Samata1, Daisuke Doi1, Kaneyasu Nishimura1, Tetsuhiro Kikuchi1, Akira Watanabe2, Yoshimasa Sakamoto3, Jungo Kakuta4, Yuichi Ono5,6 & Jun Takahashi1,7

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinsons disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specic markers such as CORIN or ALCAM has been developed, but neither marker is specic for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1 cells survive and differentiate into mDA neurons in vivo, resulting in a signicant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1 cells into the brain of an MPTP-treated monkey. Thus,

LRTM1 may provide a tool for efcient and safe cell therapy for PD patients.

1 Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. 2 Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan. 3 Group for Antibody Engineering, KAN Research Institute Inc, Kobe 650-0047, Japan. 4 Group for Seed Biologics, KAN Research Institute Inc., Kobe 650-0047, Japan. 5 Group for Neuronal Differentiation and Development, KAN Research Institute Inc., Kobe 650-0047, Japan. 6 Group for Regenerative Medicine, KAN Research Institute Inc., Kobe 650-0047, Japan. 7 Department of Neurosurgery, Kyoto University School of Medicine, Kyoto 606-8507, Japan. Correspondence and requests for materials should be addressed to J.T. (mailto:email:[email protected]

Web End =email:[email protected]).

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DOI: 10.1038/ncomms13097 OPEN

Purication of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13097

Parkinsons disease (PD) is a progressive neurodegenerative disorder caused by a loss of midbrain dopaminergic (mDA) neurons. Cell replacement therapy using human pluripo-

tent stem cells (PSCs) is expected to ameliorate the disease. A combination of dual SMAD inhibition and GSK3B inhibition has enabled us to induce mDA neurons from human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs)1,2. However, the differentiated cells are heterogeneous and may contain residual undifferentiated stem cells or proliferating neural progenitor cells, which may cause tumour formation36. Furthermore, previous clinical trials using fetal mesencephalic cells suggested that contaminating serotonergic neurons may cause graft-induced dyskinesia7,8. Therefore, the elimination of these unwanted cells is critical for clinical application in terms of safety.

For that purpose, uorescence-activated cell sorting (FACS) using antibodies for CORIN, a oor plate (FP) marker9, or ALCAM, a central nervous system microvascular endothelium marker10, have been developed. However, both CORIN and ALCAM are expressed not only in the ventral midbrain (VM) but also in more caudal FP during early brain development11,12. Therefore, cell sorting using anti-CORIN or anti-ALCAM antibodies alone cannot exclude the possibility of contaminating cells from outside the VM.

To overcome this problem, one possible strategy is to combine a rened differentiation protocol that induces mesencephalon with FACS using anti-CORIN antibody9. Another strategy is double sorting using antibodies for CORIN and OTX2, a transcriptional factor of the forebrain and midbrain13. In that study, an OTX2::GFP knock-in (KI) mouse ESC (mESC) line was established and made to differentiate into neurons. CORIN OTX2::GFP cells gave rise to more mDA neurons than unsorted cells and improved abnormal motor behaviour of 6-hydroxydopamine (6-OHDA)-lesioned rats when grafted into the striatum13. Theoretically, the combination of a fore- and midbrain marker and an FP marker enables us to isolate VM cells. However, OTX2 is not a cell surface protein but a transcription factor, meaning that its antibody cannot be used in FACS in clinical settings.

To address this issue, we took advantage of LMX1A, a transcription factor for the midbrain14. We produced LMX1A::GFP KI mESC line as a tool for visualizing midbrain cells and then performed microarray analysis to identify cell surface markers specic for VM cells by comparing gene expression proles between CORIN and CORIN cells in

LMX1A::GFP cells. Here we report that a cell surface protein, LRTM1, is specically expressed in mouse fetal VM. Purication of hiPSC-derived LRTM1 cells resulted in a higher density of mDA neurons surviving in the striatum of 6-OHDA-lesioned rats than did unsorted cells and better behavioural recovery. In addition, hiPSC-derived LRTM1 cells survived well in a short-term primate study without overgrowth.

ResultsPurication of mDA progenitors from CORIN LMX1A cells.

During early development of mouse brain, LMX1A is expressed in midbrain14, whereas CORIN is expressed in FP9. Based on these ndings, we hypothesized that VM cells including mDA progenitors can be puried by a combination of these two markers (Fig. 1ac). To visualize midbrain cells, we produced an LMX1A::GFP KI mESC line. The cells were induced to differentiate into neuronal lineage by the serum-free oating culture of embryoid body-like aggregates with the quick reaggregation method (Fig. 1d)15. Although LMX1A::GFP expression was not detectable in the early differentiation stage (Fig. 1e), it became apparent on day 9 (Fig. 1f). In support of this

trend, the messenger RNA levels of Oct-4 (also known as Pou5f1) gradually decreased from day 0 (Supplementary Fig. 1a). The emergence of LMX1A::GFP cells closely paralleled the onset of not only Lmx1a, but also Corin, Nurr1 (also known as Nr4a2)

and Th (Supplementary Fig. 1be). A ow cytometric analysis revealed that the percentage of CORIN LMX1A::GFP cells reached a peak on day 9 (30.53.1% of total cells; n 3;

Supplementary Fig. 1f). Immunostaining of the cells on day 9 showed that NURR1, a marker for postmitotic DA progenitors, was expressed by CORIN LMX1A::GFP cells (Supplementary

Fig. 1g). Intriguingly, FOXA2 FP cells16,17 were enriched in CORIN LMX1A::GFP and CORIN LMX1A::GFP populations on day 9 (Supplementary Fig. 2). Comparative gene expression analysis among these populations revealed that CORIN LMX1A::GFP cells expressed signicantly lower levels of Six3, a forebrain marker, and Gbx2, a hindbrain marker, compared with CORIN LMX1A::GFP cells or

CORIN LMX1A::GFP cells (Supplementary Fig. 3a,b). On the other hand, CORIN LMX1A::GFP cells expressed signicantly higher levels of Corin, Lmx1a and Nurr1 compared with other populations (Supplementary Fig. 3ce). To determine whether CORIN LMX1A::GFP cells give rise to mature mDA neurons more efciently, we cultured CORIN LMX1A::GFP cells and unsorted cells for another 5 days for maturation (Fig. 1d). Double-labelled immunostaining revealed that CORIN LMX1A::GFP cells gave rise to mDA neurons, which expressed TH, NURR1 and dopamine transporter (DAT)

(also known as SLC6A3), more frequently than unsorted cells (Fig. 1go). These results indicate that mDA progenitors were enriched in the CORIN LMX1A::GFP population.

LRTM1 is a cell surface marker for mDA progenitors. To identify a cell surface marker of mDA progenitors, we performed microarray analyses to compare gene expression proles between the following cell populations: (1) mESC-derived CORIN

LMX1A::GFP cells versus CORIN LMX1A::GFP cells on day 9, based on the nding that the percentage of CORIN

LMX1A::GFP cells peaked on day 9 (Supplementary Fig. 1f); and (2) CORIN cells versus CORIN cells in E11.5 mouse fetal

VM, based on the nding that CORIN is expressed by actively dividing cells in the ventricular zone of E11.5 VM (ref. 9 and Supplementary Fig. 4). We chose 83 and 677 genes from the rst and the second analysis, respectively, which were expressed at higher levels in the CORIN population (Fig. 2a,b). Among these candidates, 16 genes were commonly upregulated in

ESC-derived CORIN LMX1A::GFP cells and CORIN cells in fetal mouse VM (Supplementary Data 1). We further selected genes coding a cell surface antigen and conserved in humans, leaving ve genes as candidates for a cell surface marker of mDA progenitors: annexin A2 (Anxa2), Transmembrane 4 superfamily member 1 (Tm4sf1), Folate receptor 1 (Folr1), Tachykinin receptor 1 (Tacr1) and leucine-rich repeats (LRRs) and transmembrane domains 1 (Lrtm1). A semi-quantitative reverse transcriptasePCR (RTPCR) analysis revealed that only Lrtm1 (also known as A930016D02Rik) was specically expressed in E11.5 mouse fetal VM (Fig. 2c).

LRTM1 belongs to the extracellular LRRs superfamily18. It is composed of a signal peptide, LRR amino terminus, six LRRs, an LRR carboxy terminus, a transmembrane domain and a short cytoplasmic tail containing a short stretch of acidic residues (Fig. 3a)18. As little is known about this protein, we examined the expression of LRTM1 in mouse. A semi-quantitative RTPCR analysis revealed that LRTM1 mRNA was highly expressed in the brain but weakly expressed in the eye, lung and heart of E11.5 fetal mouse (Fig. 3b). In adult mouse, LRTM1 was expressed only in the eye and heart (Fig. 3c). A double-labelled

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13097 ARTICLE

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Figure 1 | Purication of mDA progenitors by co-expression of CORIN and LMX1A::GFP. (a) Schematic diagram of CORIN and LMX1A expression during early development of mouse. (b,c) Immunohistochemical images for CORIN (green) and LMX1A (red) in coronal and sagittal sections of E11.5 fetal mouse. Scale bars, 50 mm (b) and 200 mm (c). (d) Schematic diagram of neuronal differentiation from mESCs. (e,f) LMX1A::GFP expression of the serum-free oating culture of embryoid body-like aggregates with the quick reaggregation (SFEBq)-cultured mESC aggregates on day 2 (e) and day 9 (f). Scale bars, 200 mm. Insets indicate bright-eld images of the aggregates. (go) Immunouorescence images of the cells from unsorted and CORINLMX1A::GFP cells for TUJ1 (green), NURR1 (green), DAT (green), TH (red) and 40, 60-diamidino-2-phenylindole (DAPI; blue) on day 14. Scale bars, 70 mm. Quantication of TUJ1 TH, NURR1TH and DAT TH cells in unsorted cells (n 4) versus CORIN LMX1A::GFP cells (n 4) on day 14. Asterisks indicate

statistical signicance as determined by Students t-test, *Po0.05 and ***Po0.001. Error bars indicate s.e.m.

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Figure 2 | Gene expression proles between CORIN and CORIN populations from mESC-derived LMX1A::GFP cells or fetal mouse VM.(a) Comparison of gene expression proles between CORIN and CORIN cells in LMX1A::GFP cells on day 9. Scale bars, 250 mm. (b) Comparison of gene expression proles between CORIN and CORIN cells in E11.5 fetal mouse VM. Scales bars, 50 mm. (c) Semi-quantitative RTPCR analysis of Otx2,

Corin, Anxa2, Tm4sf1, Folr1, Tacr1, Lrtm1 and Gapdh in E11.5 fetal mouse brain.

immunouorescence study showed that the expression of LRTM1 was restricted to the VM during E10.5 to E11.5. Its expression area was overlapped with that of LMX1A, but LRTM1 was more laterally expressed (Fig. 3dk,p). On E12.5, a stage when mature DA neurons are generated, the expression of LRTM1

disappeared, whereas LMX1A was still expressed (Fig. 3lo). In E10.5, the expression of LRTM1 was also overlapped with that of FOXA2, an FP marker (Fig. 3q).

In support of these results, a ow cytometric analysis revealed that LRTM1 cells were more abundantly contained in mouse

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13097

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Figure 3 | LRTM1 is selectively expressed in VM during early brain development. (a) Schematic construction of LRTM1. (b) In E11.5 fetal mouse, LRTM1 mRNA was detected in the brain, eye, lung and heart. (c) In adult mouse, LRTM1 mRNA was detected in the eye and heart only.(do) Immunohistochemical images of fetal mouse midbrain for 40, 60-diamidino-2-phenylindole (DAPI; blue), LMX1A (red) and LRTM1 (green). Insets indicate magnied images of LMX1A DA progenitors. Scale bars, 150 mm. (p) Immunohistochemical image of E10.5 fetal mouse for LRTM1 (green),

LMX1A (red) and DAPI (blue) in the sagittal section. Inset indicates magnied images of LMX1A DA progenitors. Scale bar, 400 mm.(q) Immunohistochemical image of E10.5 fetal mouse for LRTM1 (green), FOXA2 (red) and DAPI (blue) in the coronal section. Inset indicates magnied images of FOXA2 FP cells. Scale bar, 150 mm. (r) Quantication of LRTM1 cells in E11.5 VM (n 4) versus E13.5 VM (n 4). (s) Quantication of

immunoreactive cells in the hindbrain stained for anti-Corin (n 4) versus anti-LRTM1 antibodies (n 4). Asterisks indicate statistical signicance as

determined by Students t-test, *Po0.05 and **Po0.01. Error bars indicate s.e.m.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13097 ARTICLE

VM on E11.5 than on E13.5 (Fig. 3r). Furthermore, less LRTM1 cells were contained in E11.5 mouse hindbrain compared with

CORIN cells (Fig. 3s).

These results indicate that the expression of LRTM1 in the developing brain is restricted in terms of the developmental stage, the time of emergence of mDA progenitors and location, namely the VM.

Enrichment of mouse mDA progenitors by LRTM1 sorting. To determine whether sorting with anti-LRTM1 antibody contributes to the enrichment of mDA progenitors, we dissociated E11.5 mouse VM tissue and puried LRTM1 cells by FACS.

The LRTM1 population contained more FOXA2 LMX1A mDA progenitors compared with an unsorted population (42.61.7% versus 11.11.6%; n 4 for each group;

Supplementary Fig. 5).

When we induced DA differentiation by the serum-free oating culture of embryoid body-like aggregates with the quick reaggregation method, transient expression of Lrtm1 was observed in mESC/iPSC lines (Supplementary Fig. 6a). At 9 days after differentiation of the mouse iPSC (miPSC) line 440A3, we found that B10% of total cells were LRTM1 and puried them by FACS. These cells contained more FOXA2 LMX1A mDA progenitors compared with unsorted cells (77.22.1% versus42.51.6%; n 5 and 6, respectively; Supplementary Fig. 6bd).

To determine whether miPSC-derived LRTM1 cells can survive in vivo, we puried the LRTM1 cells by FACS on day 9 and 2 days later we injected them into the striatum of 6-OHDA-lesioned rats. A large number of TH neurons survived (1,922691 cells per graft; n 3) and extended their neurites

into the host striatum at 6 weeks after transplantation (Supplementary Fig. 6e).

These results indicate that mDA progenitors can be isolated from mouse embryo and mouse PSCs by FACS using anti-LRTM1 antibody.

Enrichment of human mDA progenitors by LRTM1 sorting. To determine whether the same strategy can be applied to human mDA progenitors, we induced DA neurons from hESCs (Kh-ES1; ref. 19) and hiPSCs (1039A1; ref. 20) as previously reported (Fig. 4a)11. A comparative temporal gene expression analysis revealed that the expression of a pluripotent cell marker (OCT4) gradually decreased, whereas that of a basal and FP marker (FOXA2) and a midbrain marker (LMX1A) reached a plateau on days 714 (Supplementary Fig. 7ac). Markers for more mature DA neurons (NURR1 and TH) gradually increased for 35 days and the expression of LRTM1 peaked on day 14 (Supplementary Fig. 7df).

Based on these results, we puried human PSC-derived LRTM1 cells on day 14. An immunouorescence study of the spheres 12 h after sorting revealed that FOXA2 LMX1A mDA progenitors were more abundantly contained in LRTM1 populations compared with unsorted ones (hESC: 89.51.5%

versus 75.94.5%; hiPSC: 86.72.6% versus 72.01.3%; n 6

for each group; Fig. 4b,c). We continued culture of the spheres and performed triple-labelled immunostaining on day 28. Midbrain DA neurons expressing TH, FOXA2 and NURR1 were more frequently observed in LRTM1 populations compared with unsorted and LRTM1 populations (Fig. 4di). Further analysis of the spheres revealed that neurons expressing TUJ1 were more frequently observed in the LRTM1 population compared with the unsorted and LRTM1 populations, whereas neural precursor cells expressing NESTIN were more frequently observed in the unsorted and LRTM1 populations (Fig. 4j,m,n).

We recently reported that in a transplantation of iPSC-derived

early neural cells, SOX1 PAX6 KI67 cells form rosettes in the grafts and contribute to graft expansion21. In the present work, we found that the percentage of KI67 proliferating cells was signicantly lower and SOX1 PAX6 KI67 cells were almost eliminated in the LRTM1 population (Fig. 4k,o,p).

There were no PSCs expressing OCT-4 and only few serotonergic neurons expressing 5-HT in the spheres (Fig. 4l,q). On day 70, the LRTM1 cells expressed PITX3, a marker for mature mDA neurons22,23, and DAT, a principal regulator of DA neurotransmission (Fig. 4r,s). These cells exhibited action potentials according to electrophysiological analysis (Fig. 4t). DA levels in LRTM1 cell cultures were at least six times higher than in unsorted and LRTM1 cell cultures on day 42 (1.90.3,0.30.1 and 0.20.0 pg ml 1; n 5 for each group; Fig. 4u).

These results indicate that mDA progenitors can be isolated from human PSCs by FACS using anti-LRTM1 antibody, and that LRTM1 cells differentiate into mature mDA neurons in vitro.

Human LRTM1 cells differentiated into mDA neurons in vivo. To investigate the survival and proliferation of LRTM1 cells in vivo, we transplanted unsorted, LRTM1 and LRTM1 cells(1.3 105 cells in 2 ml for each condition) into the striatum of

6-OHDA-lesioned rats on day 28. Immunostaining for SC-121, a human cytoplasmic marker, at 12 weeks revealed that the grafted cells survived in the rat brain. The size of the LRTM1 cell-derived graft was signicantly smaller (Fig. 5ad) and consistently the number of surviving human cells (HNA cells) was signicantly lower than that of unsorted cells and LRTM1 cells (Fig. 5e). In spite of these observations, the number of TH DA neurons was higher in the LRTM1 cell-derived grafts compared with those of unsorted or LRTM1 cells (44,7777,203 versus 11,5044,689 versus 5,6322,919 cells; n 12, 7 and 5,

respectively; Fig. 5f,g,m). Furthermore, LRTM1 cell-derived grafts extended neuronal bres more extensively into the host brain (Supplementary Fig. 8). Accordingly, the percentage of DA neurons among surviving human cells was highest in the LRTM1 cell-derived grafts (29.02.6% versus 4.22.1%

versus 0.30.2%, n 12, 7 and 5, respectively; Fig. 5n). In

addition, 58.3 and 32.4% of these neurons co-expressed GIRK2 (also known as KCNJ6), an A9 DA neuron marker (Fig. 5h), or CALBINDIN, an A10 DA neuron marker (Fig. 5i). To elucidate graft components other than the mature mDA neurons, we performed immunostaining to detect early mDA progenitors (FOXA2; Fig. 5j) and postmitotic mDA progenitors (NURR1; Fig. 5k). The percentages of FOXA2 and NURR1 cells among surviving human cells were highest in the LRTM1 cell-derived grafts compared with those derived from unsorted and LRTM1

cells (FOXA2 /HNA : 76.44.6% versus 26.44.9% versus2.00.6%; NURR1 /HNA : 48.13.7% versus 17.14.5% versus 20.312.2%; n 12, 7 and 5, respectively; Fig. 5o,p).

Taken together, in the LRTM1 cell-derived grafts, B80% of the cells were in the lineage of mDA neurons, in which 30% became mature mDA neurons and the remaining 70% were still in the stage of progenitors. We also found a small amount of GFAP

HNA cells in the grafts, indicating the presence of donor-derived astrocytes (Supplementary Fig. 9). Proliferating human cells were fewer in the LRTM1 cell-derived grafts (1.80.5%

versus 4.30.6% versus 3.81.4%, n 12, 7 and 5, respectively;

Fig. 5l,q), in which most of the KI67 cells also expressed FOXA2, indicating that they were early mDA progenitors (Supplementary Fig. 9). 5-HT serotonergic neurons were hardly observed in the grafts (o0.3%).

Next, to investigate the function of LRTM1 cells in vivo, we transplanted unsorted and LRTM1 cells (1.3 105 cells in 2ml for

each condition) into the striatum of 6-OHDA-lesioned rats on day 28.

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13097

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Figure 4 | Human LRTM1 cells generate mature mDA neurons in vitro. (a) Diagram of neuronal differentiation from human PSCs (hPSCs).(b) Immunouorescence image of a hESC-derived LRTM1 sphere on day 14 for FOXA2 (green), LMX1A (red) and 40, 60-diamidino-2-phenylindole (DAPI;

blue). Scale bars, 100 mm. (c) Quantication of FOXA2 LMX1A cells in unsorted cells versus LRTM1 cells on day 14 (hESC: n 6; hiPSC: n 6).

(df) Immunouorescence images of spheres from unsorted cells, LRTM1 cells and LRTM1 cells on day 28 for NURR1 (green), TH (red), FOXA2 (white) and DAPI (blue). Scale bars, 100 mm. (g) Immunouorescence images of a sphere from LRTM1 cells for FOXA2 (green), NURR1 (green), TH (red)

and DAPI (blue) on day 28. Scale bars, 50 mm. Quantication of THFOXA2 (h) and THNURR1 cells (i) in unsorted cells (hESC: n 4; hiPSC: n 3)

versus LRTM1 cells (hESC: n 4; hiPSC: n 3) versus LRTM1 cells (hESC: n 3; hiPSC: n 3). (jl) Immunouorescence images of spheres from

unsorted and LRTM1 cells for NESTIN (green), SOX1 (green), TUJ1 (red), PAX6 (red), 5-HT (red), KI67 (white) and DAPI (blue) on day 28. Scale bars, 50 mm. Quantication of NESTIN (m), TUJ1 (n), KI67 (o), SOX1 PAX6 (p) and 5-HT cells (q) in unsorted cells (hESC: n 5; hiPSC: n 4) versus

LRTM1 cells (hESC: n 5; hiPSC: n 4) versus LRTM1 cells (hESC: n 5; hiPSC: n 4). (r,s) Immunouorescence images of LRTM1 cells for PITX3

(green), DAT (green), TH (red) and DAPI (blue) on day 70. Scale bars, 10 mm. (t) Current clamp recordings of induced action potentials by brief current pulses from human ES-derived DA neurons on day 70. (u) Levels of DA in hESC-derived unsorted (n 5) versus LRTM1 (n 5) versus LRTM1 (n 5)

cultures on day 42. Asterisks indicate statistical signicance as determined by Students t-test, *Po0.05 and ***Po0.001 (c), and a one-way analysis of variance with Bonferronis multiple comparison test, *Po0.05, **Po0.01 and ***Po0.001 (h,i,mp,u). Error bars indicate s.e.m.

Behavioural analysis showed signicant motor improvement of apomorphine- and methamphetamine-induced rotational asymmetry in both the unsorted and LRTM1 groups at 16 weeks after transplantation (Fig. 5r,s). An immunouorescence study of these rats revealed that the number of TH DA neurons was higher in the

LRTM1 cell-derived grafts compared with those derived from unsorted cells (11,7022,566 cells versus 1,102349 cells; n 7 for

each group; Supplementary Fig. 10). However, there was no clear

correlation between the number of TH neurons and the behavioural recovery (Supplementary Fig. 10b).

Finally, we examined the survival and differentiation of hiPSC-derived LRTM1 cells in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgus monkeys. LRTM1 cells were sorted on day 14 and the cultured spheres (1.0 106 cells in 4 ml) were injected into the putamen on

day 15, 21, 28 or 35. Three months after transplantation, the

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Figure 5 | Human LRTM1 cells generate functional DA neurons in vivo following transplantation. (ac) Immunouorescence images of the graft for SC-121 (green) and TH (red) at 12 weeks. Scale bars, 1 mm. (d,e) Quantication of graft volumes and HNA cells in unsorted cells (n 7) versus

LRTM1 cells (n 12) versus LRTM1 cells (n 5) at 12 weeks. Immunouorescence images of a graft containing unsorted cells (f) and LRTM1 cells

(g) for 40, 60-diamidino-2-phenylindole (DAPI; blue), HNA (green) and TH (red) at 12 weeks. Scale bars, 100 mm. (hl) Immunouorescence images of a graft containing LRTM1 cells for GIRK2 (green), CALBINDIN (green), FOXA2 (green), NURR1 (green), HNA (green), TH (red), KI67 (red) and DAPI (blue) at 12 weeks. Scale bars, 25 mm. Quantication of TH (m), TH HNA (n), FOXA2 HNA (o), NURR1HNA (p) and KI67 HNA (q) cells in unsorted cells (n 7) versus LRTM1 cells (n 12) versus LRTM1 cells (n 5) at 12 weeks. Asterisks indicate statistical signicance as determined by a

one-way analysis of variance (ANOVA) with Bonferronis multiple comparison test, *Po0.05, **Po0.01 and ***Po0.001. Error bars indicate s.e.m. (r,s) Quantication of motor behavior of 6-OHDA-lesioned rats for 16 weeks posttransplantation. (r) Methamphetamine-induced rotation (control: n 8;

unsorted: n 7; LRTM1 : n 7) was performed every 4 weeks after transplantation. (s) Apomorphine-induced rotation was performed every 8 weeks

after transplantation (control: n 7; unsorted: n 7; LRTM1: n 7). Asterisks indicate statistical signicance between control and LRTM1 as

determined by a two-way ANOVA with Bonferronis multiple comparison test, *Po0.05 and ***Po0.001. Dagger indicates statistical signicance between control and unsorted as determined by a two-way ANOVA with Bonferronis multiple comparison test, wPo0.05.

grafted cells were recognized by hematoxylin and eosin (HE)-staining and immunostaining for SC-121 (Fig. 6a,b). In the case of the day 15 cells (1 day after cell sorting), only few cells survived in the brain. Among the other conditions, the largest number (B2.4 105) of TH cells was observed in the grafts derived

from day 28 cells (Fig. 6c) and these cells extended TH neuronal bres into the host brain (Fig. 6d). Furthermore, most of them co-expressed FOXA2, NURR1 and PITX3 (Fig. 6eg), and some of these cells, were large in size and expressed DAT (Fig. 6h) and GIRK2 (Fig. 6i), indicating they were A9 mDA neurons. A small amount (o0.5%) of the cells still expressed

KI67 , which, similar to the above, also expressed FOXA2 (Fig. 6j), indicating they were early mDA progenitors. No 5-HT serotonergic neurons were observed in the graft. These results suggested that hiPSC-derived LRTM1 cells efciently survived and differentiated into mature mDA neurons in primate brain.

DiscussionIn this study, we employed a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a novel cell surface marker for mDA progenitors, LRTM1. Compared with unsorted cells, hiPSC-derived LRTM1 cells differentiated into mDA neurons more efciently in vitro. When grafted into the brains of 6-OHDA-lesioned rats and an MPTP-treated monkey, the sorted LRTM1 cells differentiated into functional mDA neurons and contained few proliferating or serotonergic cells.

LRTM1 belongs to the extracellular LRRs superfamily18. LRRs are highly versatile and evolvable proteinligand interaction motifs found in a large number of proteins with diverse functions. Especially those with extracellular LRRs are involved in various aspects of nervous system development, including axon guidance, target selection and synapse formation24,25. LRTM1 has a

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Figure 6 | HiPSC-derived LRTM1 cells survived and differentiated into mature DA neurons in primate PD model. (a) HE staining of the grafts containing LRTM1 cells at 12 weeks after transplantation. Scale bar, 500 mm. (b) Immunouorescence images of the graft derived from day 28 LRTM1 cells for SC-121 (blue), FOXA2 (green) and TH (red) at 12 weeks after transplantation. Scale bars, 500 mm. (c) Quantication of graft volume and TH cells in the grafts derived from day 21, day 28 and day 35 LRTM1 cells at 12 weeks after transplantation (n 2 for each day). (d) DAB staining of a graft derived

from day 28 LRTM1 cells for TH at 12 weeks after transplantation. Scale bars, 200 mm. (ej) Immunouorescence images of a graft containing LRTM1 cells for FOXA2 (green), NURR1 (green), PITX3 (green), DAT (green), GIRK2 (green), TH (red), KI67 (red) and 40, 60-diamidino-2-phenylindole (DAPI; blue) at 12 weeks after transplantation. Scale bars, 30 mm (e, f), 15 mm (gi) and 50 mm (j).

predicted PSD-95/Discs-large/ZO-1 domain-binding sequence at its C terminus, suggestive of synaptic location18. LRTM1 is similar to LRRTM1, which has ten extracellular LRRs and is expressed in mouse brain during early development26. LRRTM1 also contains a PSD-95/Discs-large/ZO-1 domain-binding sequence at its C terminus25. Furthermore, it binds to the presynaptic adhesion molecule neurexin2729, suggesting a role in synapse formation.

A previous study using transgenic mESC reporter lines concluded that the NURR1 stage is best for the survival of

ESC-derived DA neurons30, whereas other reports have shown that DA progenitors are enriched by sorting cells that express CORIN11 or ALCAM12. NURR1 is a transcription factor expressed by postmitotic mDA progenitors in the intermediate and mantle zones of the developing VM and also by mature mDA neurons31,32. On the other hand, CORIN is expressed by earlier mDA progenitors in the ventricular zone of

the developing VM9,32. Consistent with these previous reports, we conrmed that NURR1 was expressed by CORIN LMX1A::GFP cells (Supplementary Fig. 1g) in the differentiated LMX1A::GFP KI ES cells on day 9. These results suggest that early mDA progenitors can be sorted by using anti CORIN and LRTM1 antibodies. Both CORIN and ALCAM were expressed not only in the VM, but also in the caudal FP in the E11.5 mouse brain (Supplementary Fig. 4). In addition, ALCAM was also expressed in dorsal midbrain. In contrast, the expression of LRTM1 was restricted to the VM (Fig. 3d-s). More importantly, the expression was observed only during E10.5 and E11.5, which is when DA progenitors emerge in the VM12,33,34. ALCAM was identied by microarray analysis using E12.5 mouse brain12, which was when the expression of LRTM1 almost disappeared (Fig. 3n). These ndings indicate that LRTM1 is a more selective marker for early mDA progenitors in terms of time and localization.

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In a behavioural evaluation using 6-OHDA-lesioned rats, signicant improvement of abnormal behaviour was observed in rats that received either unsorted and cultured LRTM1 grafts (Fig. 5r,s). This nding is consistent with ours11 and other reports1,2,35,36. An immunouorescence study of the rats after the behavioural analysis revealed that the number of TH DA neurons in the grafts of unsorted cells was 1,102349.

Consistently, a previous study showed that o500 surviving TH cells derived from hESCs are sufcient to exert a behavioural effect35. In this study, we could not observe a clear correlation between the number of TH neurons and the behavioural recovery in a methamphetamine-induced rotational analysis. However, it was revealed that even 319 TH cells could lead to the behavioural recovery of 6-OHDA-lesioned rats (Supplementary Fig. 10), but this was not true for other rats. These results suggest that the behavioural recovery can be attributed not only to the number of surviving TH neurons but also possibly to the quality of TH neurons, neurite extensions from the grafts and the condition of the host environment.

Furthermore, there was a large difference in the number of surviving TH neurons between 12 and 16 weeks posttransplantation. This difference might be due to the different timing of killing (12 versus 16 weeks) or variability in the donor cells such as their differentiation stage and viability in each cell preparation. In the LRTM1 cell-derived grafts, the percentage of TH DA neurons per total surviving cells at 12 weeks was 29.02.6%, which was larger than those of

CORIN cells (18% at 16 weeks) or ALCAM cells (1.5% at 4 weeks). In addition, the percentage of 5-HT serotonergic neurons to total surviving cells at 12 weeks was 0.250.14%, which was smaller than that of CORIN cells (1% at 16 weeks)

or ALCAM cells (0.3% at 4 weeks). These results suggest that the sorting of LRTM1 cells is more effective for preparing DA neuron-enriched grafts.

In a transplantation of LRTM1 cells (1.0 106 cells in 4 ml)

into the putamen of an MPTP-treated monkey, 42 105 TH

FOXA2 DA neurons survived at 3 months, which is a number adequate for expected improvement of Parkinsonian symptoms of the clinical patient37. In addition, these neurons extended TH neuronal bres into the host brain and some of them were large in size and expressed DAT and GIRK2, suggesting mature

A9 mDA neurons. Intriguingly, the largest number of TH cells that survived occurred when the sorted cells were injected after incubation for 2 weeks. This result may be due to the damage caused by the sorting procedure (day 15 cells and day 21 cells) or excessive differentiation towards mature DA neurons (day 35 cells). Optimization of the culture condition and culture duration is needed for better outcomes.

Another advantage of sorting LRTM1 cells is the elimination of unwanted cells such as PSCs and uncommitted neural progenitors. A recent report showed that SOX1 PAX6 KI67 early neural cells form rosettes in the grafts and contribute to graft expansion21. As shown in Fig. 4j,k,m,o,p, LRTM1 cell-derived spheres on day 28 contained less NESTIN or

SOX1 PAX6 KI67 cells compared with those derived from unsorted or LRTM1 cells. In addition, LRTM1 cell-derived grafts in rat brain were smaller and contained less KI67 cells (Fig. 5ae,l,q). These results suggested that uncommitted neural progenitors (NESTIN KI67 and SOX1 PAX6 KI67 )

might cause the large graft volume in vivo, and that the sorting of LRTM1 cells reduces this risk.

In conclusion, a novel cell surface marker for mDA progenitors, LRTM1, can provide a powerful tool for efcient and safe cell therapy for PD patients. Although the current sorting yield (about 5%) is acceptable for clinical use, higher yields are preferred. New technologies, such as a more efcient

DA differentiation protocol and a gentler and faster cell sorter, should help raise this percentage.

Methods

Production of LMX1A::GFP KI mESC line. The Lmx1a-GFP targeting vector was assembled using the ploxP-GFP-neo-DT-A vector that contains GFP complementary DNA and lox-P anked HSV TK-Neomycin gene cassettes in a Bluescript SK (Stratagene) backbone. A 3.5 kb 50-arm-containing genomic fragment just upstream to the initiation codon of Lmx1a and a 3.9 kb 30-arm fragment were amplied by PCR and cloned separately into the NotI/SmaI and EcoRV sites of ploxP-GFP-neo-DT-A vector to generate the Lmx1a-targeting vector. LMX1A::GFP KI ESCs were generated by homologous recombinationin 129SVEV ESC lines according to standard procedures and homologous recombination was conrmed by Southern blotting.

Maintenance and neural differentiation of mESCs/iPSCs. mESC lines(EB5 (ref. 38); passages 3545, LMX1A::GFP KI ESCs; passages 11-21, G4-2(ref. 38); passages 2030) and iPSC line (440A-3, a kind gift from Dr Okita, Kyoto University Center for iPS Cell Research and Application, Kyoto, Japan; passages 1525) were maintained on mitotically inactivated mouse embryo broblast feeder layer in knockout DMEM medium supplemented with 1% penicillin/streptomycin (P/S; Gibco), 20% fetal bovine serum (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (2-ME; Wako), 2 mM L-glutamine (L-Glu; Sigma-Aldrich), 2,000 U ml 1 LIF (Merck Millipore) and 1 Nucleosides (Merck Millipore). We changed the

medium every day.

For neural induction, mESCs and miPSCs were replated in low cell adhesion 96-well plates (Lipidure-Coat Plate A-96U; NOF Corporation) at a density of 9,000 cells per well in a differentiation medium containing Glasgow minimum essential medium (GMEM) (Gibco) supplemented with 5% KSR, 0.1 mM MEM non-essential amino acids solution (Gibco), 2-ME, 1 mM sodium pyruvate solution (Pyruvate; Sigma-Aldrich) and 2 mM L-Glu. Moreover, we added both100 ng ml 1 FGF8b (R&D) and SHH (R&D) to induce midbrain and FP cells, respectively, from day 1 to day 6. On day 7, we added 200 nM Ascorbic acid (AA;

Nacalai), 20 ng ml 1 brain-derived neurotrophic factor (BDNF) (R&D), 1 N-2

supplement (Gibco) and removed 5% KSR. We changed the medium every 2 days.

Maintenance and neural differentiation of hESCs/iPSCs. This study was performed in conformity with The Guidelines for Derivation and Utilization of Human Embryonic Stem Cells of the Ministry of Education, Culture, Sports, Science and Technology of Japan, after approval by the institutional review board. hESCs (Kh-ES1 (ref. 19) passages 3040) and hiPSCs (1039A1 (ref. 20) passages 1525) were maintained on iMatrix-511(Nippi)-coated six-well plate at a density of 3 104 cells

per well with StemFit medium. When we began neural differentiation, these cells were dissociated into single cells with TrypLE select (Invitrogen) and were then replated on iMatrix-511-coated six-well plate at a density of 4 105 cells per well with StemFit

medium. Three days later, the medium was changed to a differentiation medium containing GMEM supplemented with 8% KSR, 0.1 mM non-essential amino acids solution, 2-ME, 1 mM Pyruvate and 2 mM L-Glu. In addition, 500 nM A83-01 (Wako) and 100 nM LDN193189 (STEMGENT) were added until day 7 and day 12, respectively; 2 mM Purmorphamine (Wako) and 100 ng ml 1 FGF8b (Wako) were added from day 1 to day 7; and 3 mM CHIR99021 (STEMGENT) was added from day 3 to day 12. We changed the medium every day11.

Cell sorting and culture. To apply FACS, cultured cells were stained with anti-CORIN antibody (1:200) or anti-LRTM1 antibody (1:20; a kind gift from Dr Ono Y., KAN Research Institute, Japan) for 30 min and the cells were then stained with Alexa 647-conjugated goat anti-mouse IgG or Alexa 647-conjugated goat anti-rat IgG antibodies (1:400; Invitrogen), respectively. Dead cells were distinguished by 7-amino-actinomycin D (BD). FACS analysis was performed using FACS AriaII (BD Biosciences) and the data were analysed by FACSDiva software (BD Biosciences). About 108 cells were applied to the sortingand B5 106 LRTM1 cells were obtained for the experiments.

The sorted cells were replated in low cell adhesion 96-well plates at a density of 2 104 cells per well and were cultured as spheres in the following neural

differentiation medium: for mouse cells from day 9 until day 11, DMEM/F12 supplemented with 1% P/S, 0.1 mM 2-ME, 200 nM AA, 2 mM L-Gln, 10 ng ml 1 glial cell-derived neurotrophic factor (GDNF), 20 ng ml 1 BDNF, 1 N2

supplement and 1 B-27 supplement; for human cells from day 14 until day 28,

Neurobasal medium (Gibco) supplemented with 1% P/S, 0.1 mM 2-ME, 200 nM AA, 2 mM L-Gln, 400 mM dbcAMP (Sigma-Aldrich), 10 ng ml 1 GDNF,20 ng ml 1 BDNF and 1 B-27 supplement. We changed the medium every

3 days and 30 mM of Y-27632 (Wako) was added in the rst medium. In the case of mouse cells, 5% KSR was also added from the day after cell sorting.

For in vitro studies of human LRTM1 cells, the cultured cells were dissociated into single cells with Accumax (Innovative Cell Technologies) on day 28 and then replated on ornithine, laminin and bronectin (OLF)-coated plates at a density of 2 105 cells per cm2. The cells were incubated in a glia-conditioned medium

consisting of Neurobasal medium supplemented with 1% P/S, 0.1 mM 2-ME,

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200 nM AA, 2 mM L-Gln, 400 mM dbcAMP, 10 ng ml 1 GDNF, 20 ng ml 1 BDNF and 1 B-27 supplement until day 70.

Histological study of mouse embryonic brain. The experiments were performed according to the Guidelines for Animal Experiment of Kyoto University, the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources (Washington, DC, USA) and the Animal Research: Reporting in vivo Experiments (The ARRIVE guidelines)39. Pregnant C57BL/6N mice (C57BL/6NCrSlc; 10 weeks old) were obtained from Shimizu Laboratory Supplies (Kyoto, Japan) and killed with pentobarbital. The embryos were removed and their brains were cut with a cryostat (CM-1850; Leica Biosystems) at 20 mm thickness and attached onto the MAS-coated slide glasses (Matsunami, Osaka, Japan). The sections containing medial midbrain were chosen for the immunouorescence study. The VM tissue was also used for the microarray and semi-quantitative RTPCR analysis.

Transplantation into the rat PD models. The experiments were performed according to the Guidelines for Animal Experiment of Kyoto University, the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources and the Animal Research: Reporting In vivo Experiments (The ARRIVE guidelines)39. Female SD rats (SpragueDawley; 9 weeks old) were obtained from Shimizu Laboratory Supplies (Kyoto, Japan). The PD models of SD rats were generated by injection of 6-OHDA (Sigma-Aldrich) into the medial forebrain bundle in the right side of the brain. The coordinates were calculated with reference to the bregma: anterior (A), 4.4 mm; lateral (L), 1.2 mm; ventral (V), 7.8 mm; and tooth bar (TB), 2.4 mm. A total of 13 mg of 6-OHDA was

injected per rat in 2.5 ml of saline with 0.02% AA. In the case of miPSCs, LRTM1 cells were sorted on day 9 and subjected to transplantation on day 11. The cultured spheres (B1 105 cells in 2 ml) in DMEM/F12 supplemented with 1% P/

S, 0.1 mM 2-ME, 200 nM AA, 2 mM L-Gln, 10 ng ml 1 GDNF, 20 ng ml 1 BDNF, 1 N2 supplement, 1 B-27 supplement and 30 mM Y-27632 were injected

stereotactically through a 22G needle into the right striatum (from the bregma: A, 1.0 mm; L, 3.0 mm; V, 5.0 mm and 4.0 mm; TB, 0 mm). A ROCK

inhibitor, Y-27632, was used to reduce dissociation-related cell death during cell transplantation40,41. In the case of hiPSCs, LRTM1 cells were sorted on day 14 and subjected to transplantation on day 28. The cultured spheres (B1.3 105 cells

in 2 ml) in Neurobasal medium supplemented with 1% P/S, 0.1 mM 2-ME, 200 nM AA, 2 mM L-Gln, 400 mM dbcAMP, 10 ng ml 1 GDNF, 20 ng ml 1 BDNF and 1 B-27 supplement, and 30 mM Y-27632 were injected stereotactically through a

22G needle into the right striatum. The rats received intraperitoneal injections of the immunosuppressant Cyclosporin A (Wako) every day starting 2 days before transplantation until the day of killing. Six, 12 or 16 weeks after transplantation, the animals were killed with pentobarbital and perfused with 4% paraformaldehyde (Wako). The brains were cut with a cryostat (CM-1850; Leica Biosystems) at 30 mm thickness and mounted. Every six sections containing the graft region were chosen for the immunouorescence study. The graft volume was calculated by identifying SC-121 positive areas in every sixth 30 mm-thick section using a uorescence microscope and the BZ-II Analyzer software program (BZ-9000; Keyence) and the total volume of the graft was determined according to Cavalieris principle.

Behavioural analysis. The methamphetamine-induced rotational behaviour was recorded for 90 min after intraperitoneal injection of methamphetamine(2.5 mg kg 1, Dainippon Sumitomo Pharma) and performed before and 4, 8,12 or 16 weeks after transplantation. The apomorphine-induced rotational behaviour was recorded for 60 min after subcutaneous injection of apomorphine(0.1 mg kg 1, Wako) and performed before and 8 or 16 weeks after transplantation. The behaviour was automatically calculated by video-monitored rotational bowls.

Transplantation into the non-human primate PD models. Two adult male cynomolgus monkeys (Macaca fascicularis; 4 years old) weighing 4.04.7 kg were provided by Shin Nippon Biomedical Laboratories (Kagoshima, Japan). The monkeys were cared for and handled according to Guidelines for Animal Experiments of Kyoto University. To generate a Parkinsonian model, the animals were given intravenous injection of MPTP HCl (0.4 mg kg 1 as a free base;

Sigma-Aldrich) twice a week until signs of Parkinsonian symptoms, such as tremor, bradykinesia and impaired balance became evident42. The coordinates of the targets were obtained from magnetic resonance images. LRTM1 cells were sorted on day 14 and subjected to transplantation on day 15, 21, 28 or 35. The cultured spheres in Neurobasal medium supplemented with 1% P/S, 0.1 mM 2-ME, 200 nM AA, 2 mM L-Gln, 400 mM dbcAMP, 10 ng ml 1 GDNF, 20 ng ml 1

BDNF and 1 B-27 supplement, and 30 mM Y-27632 were injected stereotactically

through a 22G needle into the right striatum along four tracts/side (B1 106 cells

in 4 ml per 4 injection sites per tract). LRTM1 cells at days 15 and 21 were injected into the right and left putamen of one monkey (two tracts for each condition), respectively. The cells at days 28 and 35 were injected the same way to another monkey. After surgery, the monkeys received antibiotics for 3 days and intramuscular injection of the immunosuppressant FK506 (0.05 mg kg 1, Astellas,

Tokyo, Japan) until the day of sacrice. Twelve weeks after transplantation, the animals were sacriced and perfused with paraformaldehyde under deep

anaesthesia. The brains were cut with a microtome (REM-710; YAMATO KOHKI Industrial Co., Ltd) at 40 mm thickness and mounted. Every 36 sections containing the graft region were chosen for the immunouorescence study.

Semi-quantitative RTPCR and quantitative RTPCR. Total RNA was extracted using an RNeasy Mini Kit or RNeasy Micro Kit (Qiagen) and cDNA was synthesized using Super Script III First-Strand Synthesis System (Invitrogen). Quantitative PCR reactions were performed with Ex Taq polymerase (Takara) or SYBR Premix Ex Taq (Takara), respectively, and with Thermal Cycler Dice Real Time System (Takara). The data were analysed using a delta-delta Ct method and normalized by glyceraldehyde 3-phosphate dehydrogenase levels. The primer sequences are shown in Supplementary Table 1.

Dopamine release assay. hESC-derived sorted cells on day 28 were differentiated on an OLF-coated surface for 14 days, then washed with a low KCl solution(2.5 mM CaCl2, 11 mM glucose, 20 mM HEPES-NaOH, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4 and 140 mM NaCl) and incubated in the low KCl solution for 2 min. The solution was subsequently replaced with a high KCl solution (2.5 mM CaCl2, 11 mM glucose, 20 mM HEPES-NaOH, 60 mM KCl,1.2 mM KH2PO4, 1.2 mM MgSO4 and 85 mM NaCl) for 15 min. The solution was collected in centrifugal lters (Merck Millipore) and centrifuged for 1 min at 5,200 g to remove the debris. The concentration of dopamine was detected by HPLC using a reverse-phase column and an electrochemical detector system (HTEC-500; Eicom).

Imnmunouorescence studies. For in vitro studies, the cultured cells were incubated with PBS containing 2% Triton X-100 for 30 min and then blocked with PBS containing with 4% BlockAce (Megmilk Snow Brand Co., Ltd) and 0.1% Triton X-100 for 10 min. Before blocking, the antigen retrieval procedure including heating in a microwave oven for 5 min was used for anti-SOX1 and anti-PAX6 antibodies. The primary antibody reaction was performed overnight. For in vivo studies, the brain sections were stained likewise using the free-oating method. After reaction of the primary antibodies, the samples were stained with secondary antibodies conjugated with Alexa-488, -594 and -647 (1:400, Invitrogen) or Dylight-594 (1:200, Thermo Scientic) for 30 min and then stained with200 ng ml 1 of 40, 60-diamidino-2-phenylindole. The primary antibodies are shown in Supplementary Table 2.

For 3,30-diaminobenzidine (DAB) staining, the brain slices were incubated with PBS containing 30% H2O2 for 15 min and then incubated with primary antibody overnight. After rinsing, the samples were stained with biotinylated secondary antibody (Vector Labs) for 2 h. Finally, the samples were incubated with the ABC Elite Kit (Vector Labs).

Images were visualized with a uorescence microscope (BZ-9000; Keyence) and a confocal laser microscope (Fluoview FV1000D; Olympus).

Electrophysiological analysis. Whole-cell patch-clamp recordings were carried out on 70 day cultured hESC-derived LRTM1 cells grown on an OLF-coated surface. The cells were treated with a physiological saline solution of the following composition: 2 mM CaCl2, 17 mM glucose, 2.5 mM KCl, 1 mM MgCl2, 125 mM

NaCl, 26 mM NaHCO3 and 1.25 mM NaH2PO4. Patch pipettes (GC150TF-10, Clark) had a resistance of 34 MW when lled with an internal solution composed of 0.2 mM EGTA pH 7.3, 10 mM HEPES and 140 mM KCl. Recordings with a voltage clamp and current clamp were conducted with a patch-clamp amplier (EPC-8, HEKA). The gigaseal resistances were in the range of 1020 GW. The current signals were ltered at 5 kHz through a four-pole low-pass lter with Bessel characteristics (UF-BL2, NF), sampled with a 12-bit A/D converter and imported into a 32-bit computer (PC-9821Ra333, NEC). All experiments were carried out at room temperature.

Microarray analysis. Total RNA was subjected to microarray analysis using an Ambion WT Expression Kit and Affymetrix GeneChip Whole Transcript (WT) Expression Arrays (Ambion, Life Technologies). Comparisons were performed between day 9 mESC-derived CORIN LMX1A::GFP and CORIN

LMX1A::GFP cells and between E11.5 fetal mouse VM-derived CORIN and CORIN cells. The data were analysed using the GeneSpring software programme (version 13.0; Agilent Technologies).

Statistical analysis. Statistical signicance between two samples was determined with Students t-test (GraphPad Prism 5; GraphPad). The statistical signicance among multiple samples was determined with one-way analysis of variance with Bonferronis multiple comparison tests or two-way analysis of variance with Dunnetts multiple comparisons tests. The data were considered statistically signicant for Po0.05 and are shown as the means.e. (s.e.m). All data were acquired from at least three independent experiments.

Data availability. Microarray data have been deposited in the NCBI Gene

Expression Omnibus database under accession code GSE72875. The authors

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declare that all data supporting the ndings of this study are available within the article and its Supplementary Information les or from the corresponding author upon reasonable request.

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Acknowledgements

We thank Dr N. Nakatsuji (Kyoto University, Institute for Integrated Cell-Material Sciences) for providing hESCs, Dr M. Nakagawa (CiRA, Kyoto University) for providing hiPSCs, Drs T. Yamamoto and H. Magotani (Shin Nippon Biomedical Laboratories, Ltd) for their help with the monkey, and Astellas Pharma, Inc. for FK506. We also thankDr A. Morizane, M. Motono, Mr K. Kubota, T. Sano, Y. Ioroi, Y. Miyawaki and Y. Nakajima (CiRA, Kyoto University) for their technical assistance, and Dr Peter Karagiannis for reading the manuscript. This study was supported by JSPS KAKENHI Grant Number 14J02729 (to B.S.), a grant from the Network Program for Realization of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED).

Author contributions

B.S. and J.T. designed the study and wrote the manuscript. B.S., D.D., K.N., T.K. and A.W. analysed the data. Y.O. and Y.S. generated the mESC clones. J.K. produced the LRTM1 antibody.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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How to cite this article: Samata, B. et al. Purication of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1. Nat. Commun. 7, 13097 doi: 10.1038/ncomms13097 (2016).

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