The G4–2 mouse ESC line, which constitutively expresses green fluorescent protein (GFP) under the control of the CAG promoter, was the kind gift of H. Niwa (Center for Developmental Biology, RIKEN, Kobe, Japan). Sox1-GFP knockin (46C) mouse ESCs were generously provided by Dr. Austin Smith (University of Edinburgh, Edinburgh, U.K., http://www.ed.ac.uk). The generation of 46C cells has been described previously .
To generate 46C cells expressing β-galactocerebroside (β-gal) as a graft marker, we amplified a β-geo fragment by polymerase chain reaction (PCR) from pGT1.8IRES β-geo ; the identity of the fragment was confirmed by DNA sequencing. The PCR product was then inserted into the EcoRI site of pCAGGS  to generate pCAG–β-geo. This construct was transfected into 46C cells by electroporation (250 V, 500 μF, suspended in 800 μl phosphate-buffered saline [PBS] in a 0.4-cm cuvette). Clones resistant to G418 (200 μg/ml; Sigma, St. Louis, http://www.sigmaaldrich.com) were selected. One clone (46Cβ14) expressing strong β-gal activity was used for the following transplantation experiments. All recombinant DNA research conformed to National Institutes of Health (NIH) guidelines.
Undifferentiated mouse ESCs (G4–2, 46C, and 46Cβ14) were maintained on gelatin-coated dishes in Glasgow modified Eagle's medium (GMEM; Gibco-Invitrogen, Grand Island, NY, http://www.invitrogen.com) supplemented with 1% fetal calf serum, 5% Knockout Serum Replacement (KSR; Gibco-Invitrogen), 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM pyruvate, 0.1 mM 2-mercaptoethanol (2-ME), and 2000 units/ml leukemia inhibitory factor (Gibco-Invitrogen). Mouse ESCs were differentiated in SDIA as previously reported . Briefly, ESCs were cultured on a PA6 stromal cell feeder layer in differentiation medium (GMEM supplemented with 5% KSR, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM pyruvate, and 0.1 mM 2-ME). The day on which ESCs were plated on PA6 monolayers was defined as SDIA day 0.
ESC colonies differentiated on PA6 cells for 4 days were isolated using Collagenase B (1 mg/ml; Roche, Basel, Switzerland, http://www.roche.com), dissociated into a single-cell suspension with 0.25% trypsin-EDTA (Gibco-Invitrogen), and re-suspended in cold differentiation medium. To separate two distinct cell populations, Sox-GFP+ and Sox1-GFP−, cells were sorted using a FACSAria cell sorter and FACSDiva software (Beckton, Dickinson and Company, San Jose, CA, http://www.bd.com). Dead ESCs and PA6 feeder cells were identified and eliminated by propidium iodide staining and forward-side scatter gating, respectively. Gates for each population were set so that the two subsets sorted based on Sox1 staining would not overlap when reanalyzed. Sorted cells were immediately either transplanted or replated onto chamber slides to characterize their behavior in vitro.
To examine the proliferation of the isolated cells, the sorted cells were replated onto chamber slides coated with poly-L-ornithine (Sigma), laminin (Sigma), and fibronectin (Gibco-Invitrogen) (OLF). After culture for 4 days in Alpha Minimum Essential Medium (αMEM; Gibco-Invitrogen), 5-bromo-2′-de-oxyuridine (BrdU; Nacalai Tesque, Kyoto, Japan, http://www.nacalai.co.jp) was added at a final concentration of 5 μg/ml. Twenty-four hours later, cells were fixed, denatured with 2N HCl, and stained with an anti-BrdU antibody (see below). In the differentiation assay, sorted cells were replated onto either OLF-coated slides in αMEM or PA6-coated slides in GMEM. Cells were fixed and immunostained either 5 or 10 days after replating. Nuclei were counterstained with 10 μg/ml Hoechst 33342 (Molecular Probes, Eugene, OR, http://www.probes.com).
Immunohistochemistry, Mediated dUTP Nick-End Labeling, and RT-PCR
After fixation in 4% paraformaldehyde, cells were incubated with the following primary antibodies: rabbit polyclonal antibodies against tyrosine hydroxylase (TH; Chemicon International, Inc., Temecula, CA, http://www.chemicon.com), aromatic acid decarboxylase (AADC; PROTOS Immunoresearch, Burlingame, CA, http://www.protosimmuno.com), or Ki67 (Novocastra, Newcastle upon Tyne, U.K., http://www.novocastra.co.uk), a mouse monoclonal antibody specific for Tuj1 (Covance Research Products, Richmond, CA, http://www.covance.com) and BrdU (Roche), a rat polyclonal antibody against dopamine transporter (DAT; Chemicon International, Inc.), goat polyclonal antibodies that recognize Oct4 (Santa Cruz Biotech, Santa Cruz, CA, http://www.scbt.com) or β-gal (Biogenesis, Poole, U.K., http://www.biogenesis.co.uk), and a sheep polyclonal antibody specific for TH (Chemicon International, Inc.). Appropriate cyanin-3 (Cy3)– and Cy5-labeled secondary antibodies (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) were used to visualize antibody binding. Immunostained cells and brain sections were evaluated using an Olympus DP70 optical microscope or a Fluoview FV300 laser confocal microscope (Olympus Optical Co., Tokyo, http://www.olympus.co.jp). When specified, immunostaining for Ki67 was performed using the avidin-biotin peroxidase method. Briefly, free-floating sections were incubated sequentially in rabbit anti-Ki67 antibody, biotinylated anti-rabbit immunoglobulin G (Vector, Burlingame, CA, http://www.vectorlabs.com), and avidinbiotin-peroxidase complex (Vector). Immunoreactivity was visualized using 3,3′-diaminobenzidine tetrahydrochloride dihydrate (Vector).
Cell death was determined by terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay using an In Situ Cell Death Detection Kit (Roche). TUNEL staining of both Sox1+ and Sox1− populations was performed 24 hours after plating on poly-d-lysine–coated chamber slides (Beckton, Dickinson and Company).
We extracted total RNA from both ESC colonies detached from PA6 feeder layers and FACS-sorted populations using the RNeasy Minikit (Qiagen, Hilden, Germany, http://www1.qiagen.com). FACS-sorted cells were directly collected into RLT lysis buffer. Total RNA (1 μg) was reverse-transcribed using an oligo dT12–18 primer with a Superscript kit (Gibco-Invitrogen). PCR was performed using 1/20 of the final cDNA volume with Hotstartaq DNA polymerase (Qiagen). For Sox1, Sox2, and CK17 amplification, GC melt polymerase mix (Beckton, Dickinson and Company) was used to facilitate PCR of regions with high GC content. For each amplification reaction, controls without the addition of reverse transcription (RT) were performed to exclude genomic DNA contamination. Reactions were performed at 55°C for 30 cycles, with the exceptions of Oct4 (60°C, 25 cycles) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (55°C, 25 cycles). The primer sequences and product lengths were as follows: Sox1, forward 5′-CCTCG-GATCTCTGGTCAAGT and reverse 5′-TACAGAGCCGGC-AGTCATAC, 593 bp; Sox2, forward 5′-CACAGATGCAAC-CGATGCA and reverse 5′-GGTGCCCTGCTGCGAGTA, 121 bp; Nestin, forward 5′-GGAGTGTCGCTTAGAGGTGC and reverse 5′-TCCAGAAAGCCAAGAGAAGC, 327 bp; Engrailed 1 (En1), forward 5′-TGGTCAAGACTGACTCACAGCA and reverse 5′-TCTCGTCTTTGTCCTGAACCGT, 389 bp; Oct4, forward 5′-GGCGTTCTCTTTGGAAAGGTGTTC and reverse 5′-CTCGAACCACATCCTTCTCT, 312 bp; Nanog, forward 5′-AGGGTCTGCTACTGAGATGCTCTG and reverse 5′-CAACCACTGGTTTTTCTGCCACCG, 363 bp; ERas, forward 5′-ACCATGACCCCACTATCCAA and reverse 5′-GTCT-TCTTGCTTGATTCGGC, 433 bp; CK17, forward 5′-TGC-CACCATGACCACCACCATC and reverse 5′-AGAAC-CAGTCTTCGGCATCCTT, 832 bp; GAPDH, forward 5′-GACCACAGTCCATGCCATCACT and reverse 5′-TC-CACCACCCTGTTGCTGTAG, 454 bp.
Animal experiments were performed in accordance with institutional guidelines and with the NIH Guidelines for the Care and Use of Laboratory Animals in Neuroscience Research produced by the Society for Neuroscience. All surgical procedures described below were performed after anesthesia of animals with sodium pentobarbital (30 mg/kg). Male C57BL/6 mice (Japan SLC Inc., Shizuoka, Japan, http://www.jslc.co.jp) weighing 18–22 g, which were not lesioned with either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA), were used for intracranial transplantation. For experiments in which a cell suspension was introduced into mice brains, ESC colonies formed on PA6 monolayers were detached after 4, 6, and 8 days of culture and dissociated by incubation in papain (Worthington, Freehold, NJ, http://www.worthington-biochem.com). We implanted 2 μl of a cell suspension at 105 cells per 1 μl differentiation medium or 2 × 105 FACS-sorted cells (prepared as described above) into the adult mouse striatum.
Mice were provided with drinking water containing 2% ethanol and 200 μg/ml cyclosporine A (CyA) from 3 days prior to intracranial transplantation until they were sacrificed. This treatment maintained CyA blood concentrations (measured by radioimmunoassay) at 297 ± 81 ng/ml, a level comparable to that of patients undergoing liver transplantation at Kyoto University Hospital . Under deep anesthesia, mice were placed in a stereotaxic frame (Narishige, Tokyo, http://www.narishige.co.jp) and given an injection of a 2-μl (1-μl/minutes) cell suspension into the striatum (from the bregma: A +1.0, L +2.0, V +3.0, incisor bar 0) using a Hamilton microsyringe (GL Sciences Inc., Tokyo, http://www.gls.co.jp) fitted with a 26-gauge blunt needle. Injection coordinates were determined according to the Franklin and Paxinos atlas . As a control, an additional group of mice was subjected to sham operation injecting differentiation medium alone.
Eight weeks after transplantation, mice were perfused transcardially first with PBS, then with 4% paraformaldehyde. Brains were removed and sectioned at a thickness of 40 μm. Free-floating sections were immunostained with the indicated primary antibodies and appropriate secondary antibodies as described above. The number of TH+ cells was quantified in every third section for both the graft and the surrounding tissue. These values were corrected using the Abercrombie method . The presence of grafted cells was evaluated by fluorescence of GFP, which was constitutively expressed by the transplanted G4–2 ESCs. During the grafting of cells sorted by FACS, in which GFP fluorescence was not present, hematoxylineosin (HE) staining and β-gal immunoreactivity were used to identify 46Cβ14 ESCs. The observation of a Ki67-positive mass in the brain was defined as positive tumor formation. The graft area, identified by GFP fluorescence (G4–2) or HE staining (46C), was outlined in white and examined using image analysis software (Scion Corporation, Frederick, MD, http://www.scioncorp.com). The graft volume was calculated by summing the graft areas over every sixth section (thickness, 40 μm).
To measure teratoma formation, samples at 106 cells per 10 μl differentiation medium were injected into the abdominal subcutaneous space of female CB17/Icrscid Jcl scid/scid mice (CLEA, Japan Inc., Tokyo, http://www.clea-japan.com) weighing 15–20 g. As a control, 106 naïve ESCs that had passed through the FACS machine (sham-FACS) were also injected. Resultant tumors were removed and analyzed 4 weeks later.