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Stem Cells

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Original Article Dopaminergic Differentiation of Human Embryonic Stem Cells Xianmin Zeng,a Jingli Cai,b Jia Chen,a Yongquan Luo,b Zhi-Bing You,c Erin Fotter,a Yun Wang,d Brandon Harvey,d Takumi Miura,b Cristina Backman,a Guann-Juh Chen,d Mahendra S. Rao,b William J. Freeda a

Cellular Neurobiology Branch, National Institute on Drug Abuse, bLaboratory of Neuroscience, National Institute of Aging, cBehavioral Neuroscience Branch, National Institute on Drug Abuse, dMolecular Neuropsychiatry Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA Key Words. Embryonic stem cells • Neuronal differentiation • Dopaminergic PA6 stromal cells • Hepatocyte growth factor

Abstract In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)–positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum. TH-positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for

dopamine-β-hydroxylase, a marker of noradrenergic neurons. PA6-induced neurons were able to release dopamine and 3,4-dihydroxphe-hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K+. When transplanted into 6-hydroxydopamine–treated animals, hES-derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH-positive cells were present but in very small numbers compared with the high frequency of TH-positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons. Stem Cells 2004;22:925–940

Introduction

limited availability of human cells for both basic and therapeutic research. With respect to transplantation studies, the necessity of obtaining cells from individual fetuses results in logistical problems and difficulties in minimizing variability and optimizing cells for transplantation. At least as important as therapeutic transplantation, however, is the use of

Dopaminergic neurons have potential value in cell replacement therapy, especially for Parkinson’s disease [1–3], as well as for assessing the role of potential therapeutic agents in culture assays. A major constraint in the development of transplantation therapy for Parkinson’s disease has been the

Correspondence: Xianmin Zeng, Ph.D., Development and Plasticity Section, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 333 Cassell Drive, Baltimore, Maryland 21224, USA. Telephone: 410-550-6565; Fax: 410550-1621; e-mail: [email protected] Received April 15, 2004; accepted for publication May 25, 2004. ©AlphaMed Press 1066-5099/2004/$12.00/0

STEM CELLS 2004;22:925–940

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dopaminergic neurons for in vitro research on disease mechanisms, such as neuronal degeneration in Parkinson’s disease, and the role of dopaminergic neurons in the development and maintenance of drug abuse [4–6]. If human dopaminergic neurons were readily available, studies on cellular mechanisms involved in these disorders could be greatly facilitated. Human embryonic stem cells (hESCs), derived from the inner cell mass of preimplantation embryos, can proliferate indefinitely in culture and are able to differentiate into cell types of all three germ layers in vivo and in vitro [7]. These unique properties of hESCs make them an excellent candidate as a source of functional differentiated cells for cell replacement therapies, provided that reliable means of inducing differentiation to specific cell types can be achieved. The first step to develop such cell-based therapeutics from hESCs is to define the appropriate conditions for the in vitro differentiation of hESCs into useful somatic cell types. Indeed, many clinically relevant cell types have been generated in vitro from mouse ES cells, and functional improvements have been achieved in rodent models after transplantation of dopaminergic neurons derived from mouse ES cells [8, 9]. Recently, differentiation of hESCs into multiple phenotypes, including neuronal [10–12], cardiomyogenic [13, 14], hepatic [15], pancreatic [16], and hematopoietic [17] lineages, has also been described. Although several research groups have reported the generation of neural precursors and neurons from hESCs [10–12], none of these methods produces postmitotic mesencephalic dopamine neurons at a high frequency. On the other hand, dopaminergic neurons have been efficiently generated from mouse ES cells by two different methods. One is a multiple-step method involving in embryoid body (EB) formation followed by sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8) treatment and selection for nestin-positive cells [9]. The second is a single-step method, which involves coculturing ES cells with a stromal cell line, PA6 [18]. Of the two techniques, the system of coculturing with the PA6 cell line has the advantage of simplicity and speed and can be used with cells from subhuman primates [19]. A simple and straightforward means of obtaining mature dopaminergic neurons from hESCs would be extremely valuable for both clinical application and for in vitro studies. In this study we tested whether coculture with PA6 cells can induce differentiation of hESCs and found that a high frequency of dopaminergic neurons can be generated by this method. We show that the hES-derived cells generated appear to be authentic dopaminergic neurons of the mesencephalic type. These cells synthesize dopamine and release it under physiological stimuli, and these cells can be transplanted in a rat model of Parkinson’s disease.

Dopaminergic Differentiation of hESCs

Materials and Methods Growth of hESCs The hESC line BG01 was obtained from Bresagen and cultured as described previously [20]. Briefly, hESCs were maintained on inactivated mouse embryonic fibroblast (MEF) feeder cells in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12, 1:1) supplemented with 15% fetal bovine serum (FBS), 5% knockout serum replacement (KSR), 2 mM nonessential amino acids, 2 mM L-glutamine, 50 µg/ml PennStrep (all from Invitrogen, Carlsbad, CA, http://www.invitro gen.com), 0.1 mM β-mercaptoethanol (Specialty Media, Phillipsburg, NJ, http://www.specialtymedia.com), and 4 ng/ml basic fibroblast growth factor (bFGF; Sigma, St. Louis, http://www.sigmaaldrich.com). Cells were passaged by incubation in Cell Dissociation Buffer (Invitrogen), dissociated, and then seeded at approximately 20,000 cells/cm2. Under such culture condition, the ES cells were passaged every 4–5 days.

Neural Differentiation Neural differentiation of ES cells was induced by the mouse stromal cell line PA6 as described by Kawasaki et al [18], with some modifications. hESCs were cultured to form colonies on PA6 feeder cells in Glasgow minimum essential media (Invitrogen) supplemented with 10% KSR (Invitrogen), 1 mM pyruvate (Sigma), 0.1 mM nonessential amino acids, and 0.1 mM β-mercaptoethanol. ES cell colonies were grown at a density of 1,000 colonies per 3-cm dish. The medium was changed on days 4 and 6 and every day thereafter.

Immunocytochemistry Expression of stem cell and neuronal markers was examined by immunocytochemistry, and staining procedures were as described previously [21]. Briefly, the ES cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking, the cells were incubated with primary antibody. The primary antibodies and the dilution used were as follows: Nestin and bromodeoxyurindine (BrdU [BD Pharmigen, San Diego, CA, http://www.bdscience.com], 1:500 and 1:200); neural cell adhesion molecule (NCAM), synapsin, synaptophysin, and dopamine beta hydroxylase (DBH [Chemicon, Temecula, Ca, http://www.chemicon. com], 1:200, 1:20, 1:100, and 1:200); and neuron-specific class III beta tubulin (TuJ1) and tyrosine hydroxylase (TH [Sigma], 1:2000 and 1:2000, respectively). Localization of antigens was visualized by using respective secondary antibodies (Alexa fluor 594 or 488; Molecular Probes, Eugene, OR, http://www.probes.com). For section staining, rat brains were fixed in ice-cold 4% of paraformaldehyde in phosphate-buffered saline (PBS).

Zeng, Cai, Chen et al. Cryostat sections (25 µm) were mounted onto gelatinsubbed slides. Sections were first exposed to 3% H2O2 (10 minutes) at room temperature, followed by rinsing with PBS. After blocking with 5% normal serum for 30 minutes, sections were incubated with primary antibody at 4ºC overnight. Sections were then rinsed with PBS and incubated with the appropriate secondary antibody for 1 hour before visualizing.

Reverse Transcription–Polymerase Chain Reaction Total RNA was extracted from undifferentiated or differentiated cells using RNA STAT-60 (Tel-Test Inc., Friendswood, TX). cDNA was synthesized using a reverse transcription kit (RETROscript, Ambion, Austin, TX) with 100 ng total RNA in a 20-µl reaction according to the manufacturer’s recommendations. RNase H 1 µl (Invitrogen) was added to each tube and incubated for 20 minutes at 37°C before proceeding to the reverse transcription–polymerase chain reaction (RTPCR) analysis. The PCR primers are listed in Table 1. For each PCR reaction, 0.5-µl cDNA template was used in a 50-µl reaction volume with the RedTaq DNA polymerase (Sigma). The cycling parameters were as follows: 94°C, 1 minute; 55°C, 1 minute; 72°C, 1 minute for 30 cycles. The PCR cycle was preceded by an initial denaturation of 3 minutes at 94°C and followed by a final extension of 10 minutes at 72°C. Real-time PCR was used to quantify the levels of mRNA expression of Nurr1. PCR reactions were carried out using an Opticon instrument (MJ Research, Waltham, MA) and SYBR Green reagents (Roche Molecular Biochemicals, Indianapolis) according to the manufacturer’s instructions. The content of Nurr1was normalized to the content of the housekeeping gene cyclophilin. Standard curves were generated by cloning amplified products, using human cDNA as a template, into the PCR4 vector (TOPO TA cloning kit [Invitrogen]). The purified fragment solution was measured in a spectrophotometer, and the molecular number was calculated. Plasmid solutions were then used to generate serial dilutions. PCR analyses were conducted in triplicate for each sample. The primer pairs used for real-time PCR analyses were sequence verified. The acquisition temperature for each primer pair was 3°C below the determined melting point for the PCR product being analyzed.

Detection of Dopamine BG01 cells were cultured on a PA6 cell layer for 3 weeks and rinsed twice with Hanks’ balanced salt solution (HBSS). To induce depolarization, 56 mM KCl was added into the cells for 15 minutes. The medium was then collected and stabilized with 0.1 mM EDTA and analyzed for dopamine and DOPAC.

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Dopamine and DOPAC levels were measured using an HPLC coupled to an ESA Coulochem II Detector (Model 5200, ESA, Inc., Chelmsford, MA) with a dual-electrode microdialysis cell. Data were analyzed using an ESA data station (Model 501). Samples (20 µl) were injected by an autosampler (CMA 280) into a C-18 reverse-phase column (3 µm; particle size, 3 ✕ 150 mm; Analytical MD-150 [ESA, Inc.]). The mobile phase for dopamine separation consisted of 75 mM NaH2PO4, 1.5 mM 1-octanesulfonic acid-sodium salt, 10 µM EDTA, and 7% acetonitrile (pH 3.0, adjusted with H3PO4). Dopamine and DOPAC were quantified using the reducing (–250 mV) and oxidizing electrodes (350 mV), respectively, and then calculated as nanomolar concentration. The limit of detection was approximately 0.3 pg per injection.

Focused Microarray Analysis The nonradioactive GEArrayTM Q series cDNA expression array filters for human stem cell genes pathway genes and mouse cytokine genes (Hs601 and MM-003N, SuperArray Inc, http://superarray.com) [22] were used according to the manufacturer’s protocol. The biotin 2'-deoxyuridine-5'triphosphate (dUTP)–labeled cDNA probes were specifically generated in the presence of a designed set of gene-specific primers using total RNA (4 µg per filter) and 200 U MMLV reverse transcriptase (Promega, San Luis Obispo, CA, http://www.promega.com). The array filters were hybridized with biotin-labeled probes at 60ºC for 17 hours. After that, the filters were washed twice with ✕2 standard saline citrate (SSC)/1% SDS and then twice with ✕0.1 SSC/1% SDS at 60ºC for 15 minutes each. Chemiluminescent detection steps were performed by incubation of the filters with alkaline phosphatase-conjugated streptavidin and CDP-Star substrate. Array membranes were exposed to Xray film. Quantification of gene expression on the array was performed with ScionImage software. cDNA microarray experiments were done twice with new filters and RNA isolated at different times. Results from the focused array were independently confirmed, and the array itself was validated using procedures previously described [23].

Transplantation Adult Fisher 344 rats were anesthetized with chloral hydrate (0.4 g/kg, i.p.) and injected with 6-hydroxydopamine (6OHDA; 9 µg/4 µl in normal saline containing 0.2 mg/ml ascorbic acid) over 4 minutes into the left medial forebrain bundle (–4.4 mm anterior/posterior [AP], 1.2 mm medial/lateral [ML] relative to bregma and 7.8 mm below the dura). Five weeks after lesioning, the unilaterally 6-OHDA– lesioned rats were tested for rotational behavior in response to subcutaneous methamphetamine injections (2.5 mg/kg) in an automated rotometer. Animals that showed significant


928 Table 1. Primer sequences for reverse transcription–polymerase chain reaction Gene name Tyrosine hydroxylase

Genbank accession no. NM_000360

Primer sequence 5'-TCATCACCTGGTCACCAAGTT-3' (Exon5) human specific 5'-GGTCGCCGTGCCTGTACT-3'(Exon 6/exon 7) human specific

Amino acid decarboxylase: NM_000790 dopa decarboxylase Nurr1

5'-GGGACCACAACATGCTGCTC-3' human specific 5'-CCACTCCATTCAGAAGGTGCC-3' human specific

X75918

5'-CGGACAGCAGTCCTCCATTAAGGT-3' human specific

NM_006186 trkA

Hs. 82120

5'-CTGAAATCGGCAGTACTGACAGCG-3' human specific

M23102

5'-CCATCGTGAAGAGTGGTCTC-3'

NM_002529

5'-GGTGACATTGGCCAGGGTCA-3' human specific

Hs.406293 trkB

S76473

5'-AGGGCAACCCGCCCACGGAA-3'

NM_006180 trkC

Hs.422220

5'TTGGTGGCCTCCAGCGGCAG-3'

S76475

5'-ACGCCAGGCCAAGGGTGAGCT-3'

NM_002530 GTPCH I

Hs.26776

5'-TTCATGACCACCAGCCACCAC-3' human specific

NM_000161

5'-CAGGAGACCATCTCAGATGTC-3' same for human, mouse, and rat 5'-TTCTTCTCCCTTCCCAGGCC-3' human specific

PITX3

NM_005029

5'-ACTAGGCCCTACACAC-3' human specific 5'-TTTTTTTGACAGTCCGC-3' same for human and mouse

LMX1B

NM_002316

5'-AACTGTACTGCAAACAAGACTACC-3' same for human, mouse, and rat

PCBD

NM_000281

5'-GTGATGCCATCTTCAAGCAG-3' human specific

QDPR (quinoid dihydropteridine reductase)

NM_000320

5'-AAGGAAGGACGGAACTCAC-3' human specific 5'-TCCCCAATACCAACAAATCAAC-3' human specific

DAT

L24178

5'-AGCAGAACGGAGTGCAGCT-3' same for human and rat

(SLC6A3)

NM_001044

5'-TTCATGTCCCCATCTTCATCCTC-3' similar for human and rat 5'-CAAATTAGTGTAACAGAGCCCC-3' human specific

VMAT2

Hs.406

5'-GTATGCTCTGATGCCGTCT-3' human specific

L23205

5'-CTTTGGAGTTGGTTTTGC-3' same for human and rat

NM_003054 Hs.50458

5'-GCAGTTGTGATCCATGAG-3' human specific 5'-TGCAATTGGAATGGTGGAT-3' 5'-GAGAGGGGCAAAAAGAATA-3'

DBH

NM_000787

5'-GTGCTACATTAAGGAGCTTCCAAAG-3' human specific 5'-GGCCTCATTGCCCTTGGT-3' human specific

NET

NM_001043

5'-GTAAAGTTCCTCTCGCCAGC-3' human specific

CHAT

NM_020549

5'-ATGGGGCTGAGGACAGCGAAG-3' human specific

5'-CGCGATGATAAGGAACAGTG-3' human specific Hs.302002

5'-AAGTGTCGCATGCACTGCAGG-3' human specific

VAChT

(transcript variant M)

U10554

5'-ACGTGGATGAAGCATACG-3' human specific

SLC18A3

NM_003055

5'-CTGAGACATGGCGCACGT-3' human specific

Hs.302002 GAD67

NM_000817

5'-ATTCTTGAAGCCAAACAG-3' human specific 5'-TAGCTTTTCCCGTCGTTG-3' human specific

Zeng, Cai, Chen et al.

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Table 1. (continued) Genbank accession no.

Gene name Glutaminase (KGA)

Glutaminase (GAC)

AF097494

Primer sequence 5'-GGTCTCCTCCTCTGGATAAGATGG-3' same for GAC;

NM_014905

similar for human and mouse

Hs.128410

5'-CCCGTTGTCAGAATCTCCTTGAGG-3' human specific

AF158555

5'-GATGTCCTCATTTGACTCAGGTGAC-3' human specific

Hs.128410 TPH1

NM_004179

5'-ATGATTGAAGACAATAAGGAG-3' similar for human, mouse, and rat

Hs.356479

5'-AGTTTCCATACCATCTTCCTTC-3' similar for human and rat

TPH2

NM_173353

5'-CTCTCCAAACTCTATCCCACTC-3' similar for human and rat

p75NGFR

NM_002507

5'-AGCCAACCAGACCGTGTGT-3' same for human and rat

c-RET

—

5'-GCATTCCTGTAAGCAAGTTGTC-3' human specific 5'-TTGCAGCTGTTCCACCTCTT-3' human specific 5'-AGGAGGCTGAGTGGGCTACGT-3' human specific 5'-GGACCTCAGATGTGCTGT-3' human specific c-RET

NM_000323

5'-CGACCTCATCTCATTTGCC-3' human specific 5'-AATCTTCATCTTCCGCCCC-3' human specific

GFRA1

NM_005264

5'-AGGGAAATGATCTGCTGGAGGA-3' human specific

GFRA2

NM_001495

5'-AGGCCCTGCGCCAGTTCTTCGA-3' same for human and mouse

GFRA3

NM_001496

5'-CTGCACCTCTAGCATAAGCACC-3' human specific

GFRA4

NM_022139

5'-GCTCCGAGTATGTGGCGCAGT-3' human specific

5'-CTCTGGCTGGCAGTTGGTAAAA-3' same for human, mouse, and rat 5'-ACGTTCACGTCCGTGCCGTTGC-3' human specific 5'-GGCCTTCTCGAAGAAAGTGAGC-3' human specific 5'-GAGGTCGCTGTCCTAATCAGAG-3' human specific PTCH

NM_000264

5'-TCCCAAGCAAATGTACGAGCA-3' human specific 5'-TGAGTGGAGTTCTGTGCGACAC-3' human specific

Smo

NM_005631

5'-TATTCACTCCCGCACCAAC-3' human specific

c-met

NM_000245

5'-GGAATCGAGCTGCGAGA-3' only for Arabidopsis thaliana

5'-AGCCAGACATCCAGAACTC-3' human specific 5'-TCCAACATGCAGTTTCTTGC-3' only for mouse and human

unilateral bias (>300 turns ipsilaterally) were used for transplantation studies. ES cells were cultured on PA6 cells, and colonies were isolated after 8 (n = 8) or 22 (n = 7) days of differentiation using papain dissociation kit (Worthington Inc, Lakewood, NJ) according to the manufacturer’s instructions. In addition, two animals received transplants or undifferentiated hESCs. Isolated cells were resuspended in 50% pellet volume and 50% media. Using an UMP2 syringe pump (World Precision Instruments, Inc.), an intrastriatal (0-mm AP and 2.5-mm ML relative to bregma and 6 mm below the dura) injection of 5 µl of the cell suspension was injected over 5 minutes through a 25-µl Hamilton syringe with a pulled-glass micropipette. After injection, the needle was left for an additional 2 minutes and then slowly withdrawn. Animals were euthanized for

immunostaining after 5 weeks of daily s.c. injections with 10 mg/kg of cyclosporin-A and 2 mg/kg vibramycin [24].

Results Neural Differentiation of hESCs Induced by PA6 Cells The hESC line BG01 used in this study is strongly positive for several markers of undifferentiated ES cells, such as Oct4, SSEA-4, TRA-1-60, and TRA-1-81, but negative for NCAM (neural precursor and neuron marker, data not shown). Neural differentiation of BG01 cells was initiated by culturing on a feeder layer of PA6 cells. By 6 days of culture on PA6 cells, most hES colonies had generated an outgrowth of elongated cells (Fig. 1A). By 10 days, extensive process for-

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Figure 1. Neural differentiation of hESCs induced by the stromal cell line PA6. (A–C): Phase-contrast photographs of hESCs during neural differentiation. (A): A colony with an outgrowth of elongated cells after 6 days of coculture with PA6 cells. (B): Neurite formation and typical bipolar cellular morphology at day 10. (C): By day 14, cells that migrated out of the colonies displayed bipolar or multipolar morphologies, with extensive development of fine neurites. (D–F): Immunostaining of hESC colonies after 12 days of culture on a layer of PA6 cells. (D): Nestin. (E): Neural cell adhesion molecule. (F): TuJ1. Antibody staining is in red (Nestin and TuJ1) or green (NCAM), whereas nuclear 4',6'-diamidino-2-phenylindole staining is in blue. (G): Time course of the appearance of neural markers during coculture of hESCs with PA6 cells. The percentage of colonies positive for each marker is shown as a function of the number of days cocultured with PA6 cells. NCAM first appeared on day 5, whereas TuJ1 appeared on day 8. TH-positive cells were first seen after 10 days. The scale bar is 20 µm. Abbreviations: hESC, human embryonic stem cell; NCAM, neural cell adhesion molecule; TH, tyrosine hydroxylase; TuJ1, neuron-specific class III beta tubulin.


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mation was observed on the edges of most of the hESC colonies (Fig. 1B). After 2 weeks of differentiation, cells that migrated out of the colonies formed a monolayer and displayed a bipolar or multipolar morphology characteristic of neurons, with extensive development of fine processes (Fig. 1C). Prominent fiber bundles formed by processes emanating from the colonies were frequently observed. At day 12, all hESC colonies were positive for the neural precursor marker nestin. Approximately 80% of hESC colonies contained NCAM-positive cells, and 70% of colonies were positive for the postmitotic neuronal marker TuJ1 (Figs. 1D–1F). Nestin was expressed during neural differentiation as well as in undifferentiated hESCs (data not shown). NCAM was first detected after 5 days of differentiation and increased for the next several days. NCAM peaked at day 14, at which time 92% of the colonies were NCAM positive (Fig. 1G). Likewise, colonies positive for TuJ1 first appeared at day 7, and the percentage of positive colonies increased during the following week of differentiation, so that by day 16, approximately 92% of the colonies were positive for TuJ1 (Fig. 1G). No Oct4- or SSEA-positive colonies were found after 3 weeks of differentiation on PA6 cells (data not shown).

hESCs Differentiate into Dopaminergic Neurons We next tested whether TH-positive cells can be generated from hESCs by coculturing with PA6 cells. After 3 weeks of differentiation, approximately 87% of colonies contained TH-positive cells, and a high percentage of the cells in most of the colonies were TH positive (Figs. 2A–2C). TH-positive cells first appeared between 8 and 10 days after induction and peaked at approximately 18 days (Fig. 1G). Many cells were also positive for the synaptic proteins synapsin and synaptophysin (Figs. 2D–2E). Less than 10% of colonies contained GAD65-positive cells (not shown). More than 10 cultures were immunostained for DBH and were consistently negative. No DBH immunoreactivity was seen in TH-positive cells (Fig. 2F). BrdU incorporation for 24 hours showed that after 20 days of differentiation, most cells (72%) were not dividing. Double immunostaining of TH and BrdU showed no colocalization of TH and BrdU, indicating that the TH-positive cells were postmitotic (Fig. 2G). BrdU-positive cells were usually found outside colonies or around the edges of colonies, and very few BrdUpositive cells were present within colonies. The expression of neuronal markers in PA6-induced cells was additionally analyzed by RT-PCR. Several dopaminergic markers, including TH, dopamine transporter (DAT), aromatic amino acid decarboxylase (AADC), and quinoid dihydropteridine reductase, were detected by RTPCR (Fig. 3A). Receptors such as TrkB and TrkC, GFRA1, and the Shh-mediated protein smoothed (Smo) were also

Figure 2. Dopaminergic differentiation of human embryonic stem cells. (A–C): Immunostaining of PA6-induced cells with a TH antibody after 18 days of coculture with PA6 cells. (A): A colony that contained TH-positive cells is shown. (B): A high percentage of the cells in the colonies was positive for TH. (C): Examples of individual cells that were positive for TH. (D, E): Immunostaining for the additional neuronal markers synapsin (D) and synaptophysin (E). (F): TH-positive cells (red) were negative for the noradrenergic marker DBH (green). (G): Double labeling with TH (green) and BrdU (red) antibodies after 24 hours of growth in the presence of BrdU. Numerous BrdU-positive cells were seen around the edges of the colony, but TH-positive cells were consistently BrdU negative, indicating that the TH-positive cells were postmitotic. Abbreviations: BrdU, bromodeoxyuridine; DBH, dopamine beta hydroxylase; TH, tyrosine hydroxylase.

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Figure 3. Expression of neuronal markers in hESC-derived neurons. (A): Reverse transcription–polymerase chain reaction analysis of markers of dopaminergic neurons, other neuronal markers, and stem cell markers using RNA from either undifferentiated hESCs or PA6-induced cells at day 21. (B): Quantification of Nurr1 mRNA in hESC-derived neurons and undifferentiated cells. Amplification curves corresponding to PCR4/Nr4a2 plasmid (standard curve) are shown from left to right, as follows: 18 pg/µl, 1.8 pg/µl, 0.18 pg/µl, and 0.018 pg/µl. Amplification curves corresponding to hESC-derived neurons (blue, red, and yellow curves) and undifferentiated cells (red, green, and blue curves) are represented by the first and second cluster of amplifications (from left to right; triplicates shown). Abbreviation: hESC, human embryonic stem cell.

expressed in the PA6-induced cultures but not in undifferentiated hESCs. Several markers of non-dopaminergic neuronal sub-types were also present. The cholinergic markers choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), and the glutamatergic marker glutaminase (two isoforms, GAC and KGA) were detected in differentiated cells. DBH was not detected. The transcription factors that are known to control dopaminergic differentiation, Ptx3, Lmx1b, and Nurr1, and

neurectodermal-specific transcription factor Sox1 were also detected (Fig. 3A). Most of the stem cell markers that were expressed in undifferentiated cells, including Oct4, hTERT, UTF-1, and Dppa5, were not detected or were downregulated in the differentiated cultures (Fig. 3A). Because of the importance of Nurr1 in the generation of dopaminergic neurons, we performed quantitative RT-PCR to compare Nurr1 expression in PA6-induced neurons to undifferentiated hESCs. Nurr1 transcript was detected in both


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Figure 4. Dopamine release by K+ depolarization. A representative superimposed chromatogram showing the retention time of the dopamine peak for standards (red), a sample treated with 56 mM KCl for 15 minutes (blue), and a control sample without KCl treatment (green). In most cases, no NA peak was seen; the experiment shown is the only example for which a slight possible NA peak was seen. Dopamine was not detected in the supernatant before KCl treatment or in the medium before washing with Hanks’balanced salt solution. Abbreviations: DA, dopamine; DOPAC, 3,4-dihyroxphenylactic acid; HVA, homovanillic acid; NA, novadrenalin.

undifferentiated hESCs and PA6-induced differentiated cells, but expression of the Nurr1 transcript was threefold higher in the cultures differentiated for 3 weeks (Fig. 3B). Absolute numbers of copies of the Nurr1 transcript were 8 and 24 per 1,000 copies of the cyclophilin transcript for the undifferentiated cells and PA6-induced neurons, respectively. Reverse-phase HPLC was used to examine the ability of hESC-derived neurons to release dopamine. After 3 weeks of differentiation, dopamine (2.8 ± 0.4 nM, from 107 to 108 cells) was released into the medium in response to a K+ depolarizing stimulus (Fig. 4), indicating that functional dopaminergic neurons were present in the culture. Noradrenalin (NA) was not detected in three of the four samples tested, although a small possible peak was seen in one sample, which is shown in Figure 4.

Gene Expression Profile of Differentiated Cells by a Human Stem Cell–Focused Array A focused microarray containing cDNA probes for transcripts associated with different human stem cell populations

was used to analyze gene expression in differentiated cultures compared with undifferentiated hESCs. Of the 266 genes represented by the array, 50 genes were expressed in the induced neurons but not detected in undifferentiated cells (Fig. 5, Table 2). These included 14 markers for stem and differentiated cells, 22 growth factors and receptors, adhesion molecules, and cytokines, six extracellular matrix molecules, and eight others (Fig. 5, Table 2). In particular, Sox1, Map2, TrkC, and NT3 were expressed at higher levels in the differentiated cultures, which is consistent with the results obtained by RT-PCR.

Differentiation Induced by PA6 Cells in the Presence of Growth Factors In an attempt to identify the molecular nature of the neuroninducing effect of PA6 cells, we used the MM-003N cytokine growth factor–focused array to compare PA6 cells with MEFs. In general, cytokine and growth factor expression patterns were remarkably similar for PA6 cells and MEFs. Hepatocyte growth factor (HGF) mRNA was, however, expressed at sixfold higher levels in PA6 cells than in MEFs


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Category

Genes expressed in human embryonic stem–derived neurons

ECM molecules

β-catenin, cadherin 2, cadherin 5, cadherin 15, catenin delta 2, CTNNA2

Molecular markers for stem and differentiated cells

AFP, FLK1/VEGFR2, GFAP, GP130, LIFR, MAP2, MYH6, NCAM, NCAM2, NT-3, PDGFR2, PROX1, SNAI1, SOX1

Growth factors and receptors, ACVR2,ALK-5/ACVRLK4, BMP10, BMP3, BMP5, CNTF, CNTFRa, DLK, EGF, FGF8, adhesion molecules, and cytokines FGFR3, FGFR4, FZD1, FZD2, GDF1, NEUROG1, TGF-b2, TGF-b3, TGF-b1, TrkC, WNT4, WNT11 Others

CXCR4, cyclin E1, DNMT1, p16INL4, PDX1, SDF1, SOX4, SOX17

Figure 5. Gene expression profile of neurons derived from hESCs by a focused human stem cell microarray. Images of arrays hybridized using undifferentiated hESCs and cells that had been differentiated on PA6 cells. Abbreviations: hESC, human embryonic stem cells.

(Fig. 6A). Greater expression of vascular endothelial growth factor (VEGF) and FGF7 mRNAs was also found in PA6 cells (Fig. 6A). RT-PCR confirmed that transcripts of HGF, VEGF, and FGF7 were increased in PA6 cells compared with MEFs (Figs. 6B, Table 3). To determine whether these growth factors play a role in inducing neuronal differentiation, we compared differentiation of hESCs in the presence or absence of HGF, VEGF, and FGF7 using either PA6 cells or MEFs as feeder layers. No significant difference in the number of TH-positive colonies was observed among the cells differentiated either with or without HGF, VEGF, and FGF7 or with anti-HGF when cocultured with PA6 cells (data not shown). When hESCs were grown on PA6 cell membranes or lysed PA6 cells, with or without HGF, VEGF, and FGF7, few colonies survived and neuronal differentiation was not seen.

Because bone morphogenic protein (BMP) and serum have been demonstrated to inhibit neural differentiation in mouse and primate ES cells, we also tested whether BMP and serum have a similar effect on neural differentiation of hESCs induced by PA6 cells. When BMP4 or FCS was added to the medium, less than 10% of the colonies were TH-positive after 3 weeks of differentiation, indicating that BMP4 and serum suppressed neural differentiation of hESCs.

Transplantation of PA6-Induced Cells into the Rat Brain To test whether PA6-induced dopaminergic cells derived from hESCs could be integrated in vivo, we transplanted these cells into the striatum of rats that had received unilateral lesions with 6-OHDA 4 weeks before transplantation and had been tested for amphetamine-induced rotation to


935 B: Data summary Gene names

Hgf

MEFa

PA6a

PA6/MEF

0.19

1.23

6.32

Nfkbia

0.06

0.20

3.67

Fgf7

1.35

2.05

1.52

Figf

1.65

2.05

1.24

Fgf9

0.84

0.94

1.12

Vegfb

0.49

0.28

0.57

Vegfc

0.42

0.08

0.18

Fgf2

0.58

0.09

0.15

Fgf10

0.51

0.04

0.08

Ptn

2.59

0.95

0.37

Fgf3

1.82

0.99

0.54

Ifg1

0.80

0.00

0.00

a

Data are expressed as ratio of the intensities to those of GAPDH

verify lesion completeness. Two differentiation stages were used, early neural precursors after 8 days of coculture on PA6 cells and postmitotic neurons after 22 days of coculture on PA6 cells. Two animals received transplants of undifferentiated hESCs. Immunohistochemical analysis was performed 5 weeks after grafting, and human cells were found in all brains transplanted with either 8- or 22-day cells. Some TH-positive human cells were also identified in both groups of animals (Figs. 7A–7E). Numbers of TH-positive cells were small, but a few clearly stained cells were found throughout the graft sites. In addition, larger numbers of weakly positive cells and cells with unclear morphology were also seen (Fig. 7F). An approximation of relative numbers of TH-positive cells was made by counting the total number of TH-positive cell profiles (clear cell morphology with unstained nucleus) for 10 sections from each animal (Table 3). Greater numbers of THpositive cells were found in the brains of animals transplanted with 22-day differentiated cells compared with the animals transplanted with 8-day differentiated cells. No Oct4-positive cells were seen in any of the brains transplanted with either 8- or 22-day differentiated cells or undifferentiated cells, indicating that pluripotent hESCs did not persist or expand after transplantation. Smooth muscle actin (SMA) was chosen to monitor the presence of a cell type other than neuronal ectoderm derived from hESCs in the transplanted cells. SMA-positive cells were found in all of the animals. The number of SMA-positive cells in the animals transplanted with 22-day cells was approximately half that seen in animals transplanted with 8-day cells (Figs. 7G, 7H; Table 5). Larger numbers of SMA-positive cells were found in the animals that received transplants of undifferenti-

Figure 6. Gene expression profile for PA6 cells and MEFs by a focused murine cytokine microarray. (A): Images of arrays hybridized using RNA from either PA6 cells or MEFs. (B): Reverse transcription–PCR confirmation of differentially expressed genes. Abbreviations: MEF, mouse embryonic fibroblast; PCR, polymerase chain reaction.

ated cells (Fig. 7I; Table 5). Thus, coculture with PA6 cells did not preclude the differentiation of a significant number of cells with mesodermal properties.

Discussion Generation of dopaminergic neurons at high frequency from hESCs has not previously been reported. In the present study,

936


Figure 7. Immunohistochemistry of hESCs after transplantation into the brain of cyclosporin-immunosuppressed rats with 6-hydroxydopamine lesions. (A–E): TH-immunoreactive cells double stained for anti-human antigen. TH staining is in green, and anti-human antigen is in red. Nuclei are stained with DAPI (blue). (A, B): hESCs differentiated for 8 days before transplantation. (C–E): Cells differentiated for 22 days before transplantation. (F): Low magnification of a section from an animal transplanted with cells differentiated for 22 days, showing a larger number of TH-immunoreactive cells with unclear neuronal morphology. These cells, which are triple labeled with TH (green), anti-human antigen (red), and DAPI (blue), appear white. The red cells are transplanted hESCs, which are negative for TH. (G–I): Sections are immunostained for smooth muscle actin (red), a marker for a non–ventral nervous system cells. Nuclei are stained with DAPI (blue). (G): Section from an animal transplanted with undifferentiated cells. (H): Cells differentiated for 8 days before transplantation. (I): Cells differentiated for 22 days before transplantation. Abbreviations: DAPI, 4',6'-diamidino-2phenylindole; hESC, human embryonic stem cell; TH, tyrosine hydroxylase.

we report that cells with a number of properties consistent with those of dopaminergic neurons can be efficiently generated from a hESC line, BG01, in vitro by coculture with PA6 cells. These hESC–derived neuronal cells are engraftable, and small numbers of TH-positive cells survived transplantation into the rat brain. Similar results were observed using another hESC line, H1 (unpublished results). Transplantable neural precursors have recently been isolated from hESCs by two research groups [11, 12]. Reubinoff et al. [11] used a method in which hESC colonies with cells containing short processes were manually extracted, followed by serum-free culture, whereas Zhang et al. [12] induced neural differentiation via EB formation followed by culturing in the presence of FGF2. Although these neural precursors differentiated into neuronal phenotypes in vivo and in vitro, only a few neurons expressed TH. Park and colleagues [24, 25] also recently reported that some TH-positive cells

could be generated from hESCs by a combination of factors, including brain-derived neurotrophic factor (BDNF), transforming growth factor-alpha, bFGF, and retinoic acid. Each of these methods requires multiple steps and growth factors. In contrast, the present method resulted in the differentiation of a high percentage of dopaminergic neuronal cells from hESCs, which is necessary for potential therapeutic application. Nonetheless, differentiation was not uniform, and cell types other than dopaminergic neurons were present. The number of TH-positive cells present in the brain of the transplanted animals was small, and far greater numbers of surviving transplanted cells would presumably be required to obtain functional changes in, for example, animal models of Parkinson’s disease [26, 27]. This was not simply an issue of the total number of TH-positive cells that survived after transplantation, but in addition the frequency of TH-positive cells was much lower than that seen in vitro with PA6 cell


937

Table 2. Differentiation of BG01 cells on different feeder layers or growth factors Conditions

Growth

Differentiation

Dopaminergic differentiation

Gelatin or collagen I coated

No

—

—

MEFs + HGF/VEGF/FGF7

Yes

Yes

No

MEFs + anti-HGF

Yes

Yes

No

PA6 + HGF/VEGF/FGF7

Yes

Yes

Yes

PA6 cells + anti-HGF

Yes

Yes

Yes

PA6 + BMP (with serum)

Yes

Yes

No

PA6 + BMP (without serum)

No

—

—

PA6 cells + serum

Yes

Yes

No

Lysed PA6 cells

No

—

—

PA6 cell membranes

No

—

—

Abbreviations: BMP, bone morphogenic protein; FGF7, fibroblast growth factor 7; HGF, hepatocyte growth factor; MEF, mouse embryonic fibroblast; VEGF, vascular endothelial growth factor.

coculture. Moreover, surviving non–central nervous system cells were present in the brains of transplanted animals, so that homogeneous differentiation into TH-positive cells was not achieved by this method. The number of surviving dopaminergic neurons following transplantation to the brain was small despite the fact that many thousands of dopaminergic cells were implanted. This suggests that the transplantation procedure or the host brain environment was not ideal for the survival of transplanted human dopaminergic neurons generated by the present procedure. Fundamental improvements are likely to be required to result in a functionally effective transplantation procedure. In addition, the possibility of fusion between host and donor cells [28] was not addressed in the present study. Dopaminergic neurons with slightly different properties are found in the mesencephalon, hypothalamus, olfactory bulb, and retina [29]. Dopaminergic neurons in general express particular and unique patterns of proteins and mRNA molecules. Among these markers are several unique tran-

Table 3. Mean number of cells per section positive for tyrosine hydroxylase (TH) or smooth muscle actin (SMA) Transplanted with

TH positive SMA positive

22-day cells

8.8

22.1

8-day cells

3.8

38.8

Undifferentiated cells

0.4

63.1

Numbers of cell profiles positive for each marker were counted in 10 sections per animal. Cells were counted as THpositive only if a clear neuronal morphology with a stained cytoplasm and an unstained nucleus was visible.

scription and differentiation factors, some of which promote the development of region-specific subtypes of dopaminergic neurons [30]. Consistent with this notion, the tyrosine hydroxylase promoter contains elements that are specifically activated in different brain regions [31]. Several factors can stimulate dopaminergic neuron development. Nurr1, Lmx1b, and Ptx3 transcription factors are mesencephalon specific, whereas the messenger molecules Shh and FGF8 seem to promote the dopaminergic phenotype irrespective of brain region [30, 32, 33]. Other transcription factors, including Sox1, influence neuronal specification per se [34]. Nurr1 is essential for development of the dopaminergic neurotransmitter function in mesencephalic neurons [35]. A second pathway involving Lmx1b and Ptx3 seems to be important for expression of other aspects of the dopaminergic neuronal phenotype [36]. Shh and FGF8 induce the dopaminergic phenotype when present in appropriate concentrations in specific brain regions [33, 37, 38]. The receptor for Shh is Smo, which is inhibited by a second protein patched (ptch) when Shh is absent [39–41]. There are several alternatively spliced forms of FGF8, which differentially interact with certain forms of FGF receptors FGFR2, FGFR3, and FGFR4 [42]. In addition to developmental pathways, dopamine neurons can be defined by neurotransmitter function and response to growth factors. Markers for mature dopamine neurons thus include proteins directly involved in dopamine neurotransmitter biosynthesis and function, especially TH, AADC, DHPR, GTPCH, and DAT. Dopamine neurons also respond to the growth factors BDNF and GDNF [43–46]. The receptor for BDNF is Trkb [47, 48]. GDNF and the related molecules neurturin, artemin, and persephin interact with GFRA1, GFRA2, GFRA3, GFRA4, and cRET [49, 50].

938

The expression of markers for dopaminergic neurons, as well as other neuronal markers, in hESC-derived differentiated cells was examined by immunocytochemistry, RT-PCR, and microarrays. The markers associated with the mature dopaminergic neuronal phenotype TH, DAT, AADC, GTPCH, PCD, DHPR, and VMAT2 were expressed. The growth factor receptors TrkA, TrkB, TrkC, GFRA1, GFRA2, GFRA3, p75R, and c-ret and the Shh receptors Ptch and Smo were also present. Transcription factors Nurr1, Ptx3, Lmx1b, and Sox-1 associated with dopaminergic and neuronal differentiation were expressed by the PA6 cell–induced cells. Nurr1 was detectable in both undifferentiated hESCs and PA6-differentiated cells, but quantitative RT-PCR verified that a threefold increase in expression was associated with differentiation. The reason that Nurr1 and other markers, which are generally associated with differentiation, were expressed in undifferentiated BG01 cells is unclear; however, it has been reported that hESC lines express some markers that are not expressed in mouse ES cells [51], and we cannot exclude the possibility that a small number of differentiated cells were present within the hESC colonies. DBH was not expressed in the TH-positive cells by immunostaining or RTPCR, and little or no NA was released by KCl stimulation, supporting the conclusion that PA6-induced hESC-differentiated cells are dopaminergic rather than noradrenergic. In addition to dopaminergic markers, cholinergic (ChAT and VAChT) and glutamatergic (GAC and KGA) markers were detected in the induced neurons, indicating the potential for generation of multiple neuronal types by this method. On the other hand, undifferentiated ES cell markers (hTERT, Oct3/4, Dppa5, and UTF-1) were not expressed in the differentiated cultures, indicating that undifferentiated hESCs do not persist in hESC cultures differentiated on PA6

Dopaminergic Differentiation of hESCs cells. Although SMA-positive cells were present in the brain after transplantation, indicating that differentiation on PA6 cells did not preclude mesodermal differentiation, Oct4-positive cells did not persist or appear after transplantation. This is important for potential clinical use, because even a few undifferentiated ES cells could form teratomas when transplanted. Kawasaki et al. [18] originally used the term stromal cell-derived inducing activity (SDIA) to describe the dopaminergic neuronal-inducing effect of PA6 cells. To identify the factors responsible for SDIA, a focused microarray was used to compare the expression of growth factors and cytokines between MEFs and PA6 cells. Very few growth factor messages differed between the two cell types, but the HGF transcript was sixfold higher in PA6 cells, and higher expression of FGF7 and VEGF was also seen. These growth factors alone or in combination were not, however, able to induce dopaminergic differentiation in hESCs. Therefore, PA6 cells may stimulate dopaminergic differentiation by a complex combination of growth factors, including other unidentified components. It is also possible that the supporting environment provided by PA6 cells, or an interaction between PA6 cells and hESCs, plays a role in their neuroninducing activity. Interestingly, there has been one report that HGF stimulates the survival and differentiation of dopaminergic neurons [52]. Therefore, it remains a possibility that HGF contributes to the dopaminergic neuronal-inducing effect of PA6 cells. In summary, coculture with PA6 cells is a simple and efficient method of generating postmitotic dopaminergic neuron-like cells from hESCs. Dopaminergic cells derived from hESCs are functionally and biochemically similar to normal dopaminergic neurons and survive transplantation for at least 5 weeks.

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