MEF2C Enhances Dopaminergic Neuron Differentiation of Human Embryonic Stem Cells in a Parkinsonian Rat Model

Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to treat Parkinson's disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in vitro. Conversely, RNAi knockdown of MEF2C in NPCs decreases neuronal differentiation and dendritic length. When we inject MEF2CA-programmed NPCs into 6-hydroxydopamine—lesioned Parkinsonian rats in vivo, the transplanted cells survive well, differentiate into tyrosine hydroxylase-positive neurons, and improve behavioral deficits to a significantly greater degree than non-programmed cells. The enriched generation of dopaminergic neuronal lineages from hESCs by forced expression of MEF2CA in the proper context may prove valuable in cell-based therapy for CNS disorders such as PD.

Lentiviral transfer vectors included PGK-GFP (control), PGK-MEF2CA-IRES2-GFP (lenti-MEF2CA), PGK-Bcl-xL-IRES2-GFP (lenti-Bcl-xL), shRNAs against MEF2C generated in pGIPZ-shRNAmir (shMEF2C-1 and shMEF2C-2), and non-silencing shRNA in pGIPZ-shRNAmir (scrambled shRNA control). Lentiviral constructs were prepared in house, except for shRNAs, which were obtained from a commercial source (Open Biosystems). Each lentiviral transfer vector was transduced into HEK293 cells with pCMVΔR8.74 and pMD.G to generate a replication-deficient lentivirus. On day 0, HEK293T cells were plated in 150-mm dishes with 15 x 10 6 cells/dish. On day 1, cells (~80% confluent) were transfected with three plasmids (lentiviral transfer vector, pCMVΔR8.74, and pMD.G) mixed in a ratio of 3:2:1 (30, 20 and 10 µg, respectively) and brought up to 1350 µl with H 2 O; 150 µl of 2.5 M CaCl 2 were added drop wise. Finally, 1500 µl of 2x Hanks' balanced salt solution (HBSS: 8 mg NaCl, 0.37 mg KCl, 0.19 mg Na 2 HPO 4 ·7H 2 0, 1 mg glucose, 5 mg HEPES, pH 7.2 in 500 ml total volume) were added drop wise with constant vortexing. The mixture was incubated for 15 min at room temperature and applied to the cells in 16 ml of medium. Cells were incubated for 7 h in a 5% CO 2 incubator at 37 °C. Then, the medium was replaced with viral harvesting medium (Ultraculture medium, Bio-Whittaker) and further incubated for 48 h. On day 3, the medium was collected and centrifuged for 10 min at 400 x g and filtered with 0.45 µm pore diameter, low protein binding-cellulose acetate. To concentrate virus, the filtrate was centrifuged at 20,000 rpm To measure MEF2CA activity after transduction with a lenti-MEF2CA construct, SH-SY5Y cells were plated on 24-well plates at 1 x 10 5 cells/well. The next day, each viral construct was added at a MOI of 0.5 for 7 h, washed once with PBS, and cultured for an additional day.
Cells were transfected using Fugene HD (Roche) with 0.5 µg of MEF2 response element (RE)-MHC-Luciferase plasmid and 0.05 µg of Renilla as an internal control in each well. Two days later, cells were washed, lysed with 1x Passive lysis buffer (Promega), and quantified using the Dual-Luciferase reporter assay system (Promega). To monitor the expression of GFP after infection, SH-SY5Y cells infected with a MOI of 0.5 or 1 were lysed with 1x SDS buffer without reducing agent or bromophenol blue and quantified by BCA (Pierce). Rabbit anti-GFP antibody was used for probing GFP expression.

Infection of hESC-Derived R-NSCs with Lentiviral Constructs
R-NSCs were treated with Accutase (Chemicon) to make a single-cell suspension and transferred onto low-attachment plates (24-well plate, Costar) containing 300 µl NPM/well. Each lentiviral construct was added and incubated for 5 h, at which point cells were transferred without washing onto a 6-well low-attachment plate with 1.5 ml NPM/well. The next day, infected cells were washed once with PBS and placed back onto low-attachment plates in NPM for one week to allow for expansion. Infected cells were dissociated and plated onto PLO (10 µg/ml)/LN (1 µg/ml)-coated plates in NPM, at which time they had become hNPCs in monolayer culture.
Confluent hNPCs were dissociated and replated in TDM on glass coverslips coated with PLO/LN for terminal differentiation. To assess changes over time in culture, these cells were stained with anti-GFP (to identify cells infected with control lentivirus, lenti-MEF2CA or lenti-Bcl-xL) or with anti-turbo GFP (to identify cells infected with lenti-scrambled shRNA or lenti-shMEF2Cs), and anti-doublecortin or anti-MAP2 (to identify neurons).
The neurospheres were then plated onto coverslips coated with PLO (50 µg/ml)/LN (5 µg/ml) in differentiation medium (bFGF/EGF-free neurobasal/2% B27 medium) for 3 days prior to assessment of neuronal marker expression. Alternatively, infected cells were directly plated onto PLO/LN-coated coverslips, incubated for one day in proliferation medium, and then cultured for 7 days in differentiation medium prior to measurement of neurite length; this alternative plating technique was used in order to obtain a monolayer of cells for neurite assessment. The longest neurite was picked and the length was measured by NeuronJ (an ImageJ plug-in) [3].

Enhancer/Promoter Analysis
Promoter regions, including the transcriptional start site (+1) of each gene, were retrieved using the transcriptional regulatory element database (TRED); MEF2 binding sites were predicted by PROMO using v. 8.3 of TRANSFAC software with a 10% maximum matrix dissimilarity rate.

Quantitative RT-PCR and Immunoblot Analyses
Endogenous expression of each gene of interest was assessed at different time points by qPCR using cDNA (miScript reverse transcription kit, Qiagen) obtained from total RNA (miRNAeasy mini kit, Qiagen). For qPCR, we used a LightCycler 480DNA SYBR green I master mix (Roche Applied Science) with the QuantiTect primer assay (Qiagen). The primers for endogenous

TUNEL Assay
Differentiated hNPCs infected with lenti-scrambled shRNA control or lenti-shMEF2Cs were assayed for apoptosis with the ApopTag In Situ Apoptosis Detection Kit (Chemicon) according to manufacturer's instructions with minor modifications. Precooled acetone:methanol (1:1) rather than ethanol:acetic acid (2:1) was used to post-fix cells. To identify cells infected with each virus, anti-turbo GFP antibody was added before mounting samples. Images were viewed by deconvolution microscopy using standard FITC (for turbo GFP signal), Cy3 (for signal labeled with ApopTaq), or DAPI excitation and emission filters.

Immunocytochemistry/Immunohistochemistry
Cells in culture were fixed with 4% paraformaldehyde for 10 min at 25 °C and then permeabilized with 0.25% Triton X-100 in PBS for 10 min. Parkinsonian rats were perfused with 4% paraformaldehyde, and, after removal from the cranium, brains were post-fixed for 4 h at 4 °C, then cryoprotected in 30% sucrose prior to freezing. Cryostat sections were cut at a thickness antibodies. Rhodamine Red conjugated Streptavidin (Pierce) was used for Biotin labeled antiguinea pig or -mouse IgG. Images were generated by deconvolution microscopy; filters for DAPI, CY3, CY5, and FITC were used for three or four-color image capture, and a 'nearest neighbor' analysis was employed in the quantitative deconvolution software algorithm (Slidebook, Intelligent Imaging Innovations). For photomicrographs of grafts and host neuropil in the brains of parkinsonian rats, sections were incubated with anti-tyrosine hydroxylase antibody followed by biotinylated secondary antibody (Vector) and avidin-conjugated peroxidase (Vector). Staining was visualized by incubating in diaminobenzidine (DAB) solution according to the manufacture's directions (Vector). Sections were scanned at a magnification of 20x (resolution of 0.5 µm/pixel (50,000 pix/inch)) using an Aperio ScanScope XT Automated High Throughput Scanner (Aperio Technologies). Background illumination levels were calibrated using a prescan procedure. For neuropil quantification, sections were imaged with a 60X objective using a bright field Olympus digital microscope. Parameters for images were: dimensions = 512 x 512 pixels; zoom = 3; gain = 40 -45; offset = 15 -20. DAB intensity associated with TH-immunoreactive structures was measured using NIH ImageJ software. Areas distal to the graft were imaged to provide intensity values as controls to subtract staining background for each tissue section.

Electrophysiology
To prepare cells for electrophysiological recording, R-NSCs were infected with a lenti-MEF2CA construct and kept on low-attachment plates for two weeks for cell expansion. R-NSCs were dissociated with Accutase into single cells that were then sorted into two groups (GFP+ and GFP-) using a FACSVantageSE DiVa (BD Biosciences). Sorted GFP+ cells were plated back onto low attachment plates and grown as neurospheres for two additional weeks in NPM in order to recover and expand, and then plated onto PLO (10 µg/ml)/LN (1 µg/ml)-coated dishes to become NPCs in monolayer. The GFP+/NPCs were plated onto 12-mm diameter glass coverslips were achieved within 50-100 ms, and a pipette containing bath solution was used to rapidly washout applied drugs.

Chromatin Immunoprecipitation (ChIP) Assay
ChIP assays were conducted as previously described with some modifications [4]. Briefly, about 20-30 million R-NSCs were used for each ChIP experiment. R-NSCs were dissociated with Accutase and crosslinked with 1% paraformaldehyde in PBS for 10 min at room temperature. For qPCR, primers were designed to surround a region 150-250 bp from the putative MEF2 response elements in the Etv1 or nurr1 promoter, and used to determine the enrichment of specific sequences after ChIP. This enrichment was determined by qPCR using a SYBR Greendetection kit (Invitrogen) on a Mx3000P real time PCR system (Stratagene). Levels of enrichment after ChIP were calculated using the comparative cycle threshold method (Invitrogen) after normalizing with the IgG control. Primer sequences used for qPCR were as follows: for the putative MEF2C response element in Etv1 promoter, 5'-cttgttcggatttttgctactacc-3' (forward), 5'-catagctaattaccctccgacagt-3' (reverse); for the putative MEF2 response element in nurr1 promoter, 5'-tgtgcttgagtttccgactg-3' (forward), 5'-cagcaactccagctcagagg-3' (reverse).
Immediately after injection, the animal was placed into the rotometer and the time until the first full rotation recorded. The number of turns was recorded in 5-minute intervals for 30 minutes.
Rats that had substantial deficits (>180 rotations/30 min) were divided randomly into two groups, one to receive control/R-NSCs and the other MEF2CA/R-NSCs. For stem cell transplantation, we followed previously published protocols [5,6]. Briefly, rats were transplanted with ~ 5-6 x 10 5 stem cells in 3 µl HBSS injected into the striatum, ipsilateral to the lesion (Coordinates from bregma: AP: 0, ML + 2.8 mm, at three different depths DV -6, -5, -4, as measured stereotactically). For transplantation, cells were dissociated with accutase into a single-cell suspension. Aggregates or clumps were removed by passage through a 35-µm mesh cell strainer.
Dissociated cells were quantified microscopically and viability assessed by exclusion of 0.4% trypan blue. Viability was ~90%, and the transplanted cell number was based on the live-cell count. Rats were immunosuppressed with daily intraperitoneal (i.p.) injections of 20 mg Cyclosporin A/kg (LC Laboratories) for 5 days, starting 2 days before transplantation. Thereafter, daily i.p. injections of Cyclosporin A at 15 mg/kg were given until sacrifice. Apomorphine test regimens were performed every two weeks after transplantation, following the procedures described above. Additionally, we assessed the effect of stem cell transplantation with the limbuse asymmetry (cylinder) test [7]. For this purpose rats were placed in a transparent cylinder (20 cm in diameter, 30 cm in height), and exploratory activity was recorded with a digital video camera for 10 minutes. Forelimb used in each of three behaviors was counted: initially contacting the wall of the cylinder when rearing, movements along the wall while rearing, and landing after rearing. As described previously, in each trial rats had to exhibit at least five such movements to be scored. Counts for initial wall contact and movements along the wall were combined for analysis. Asymmetry scores were calculated by the formula: (%Mn = percent ipsilateral forelimb movements, %Mi = percent contralateral forelimb movements) [7].

Quantification of DA Neurons
Lenti-MEF2CA expressing hESCs were differentiated and plated under terminal differentiation conditions. Cells were fixed at Neural Stage I for rabbit anti-GFP and mouse anti-TH antibody staining, or at Neural Stage II for rabbit anti-GFP and mouse anti-EN antibodies staining. Two independent experiments were carried and five different fields from each experiment were quantified. Over 1200 cells were counted for TH+ neurons and over 500 cells were for EN+ cells.
For quantification of TH+ neurons from PD rats, brains were prepared and sectioned as described above (see Immunocytochemistry/Immunohistochemistry section). Three random animals were chosen from both the control and MEF2CA groups, and ten random fields were selected in multiple sections at the same distance from the Bregma for each rat. Each section was co-stained with rabbit anti-GFP and mouse anti-TH antibodies, and visualized with Alexa 488 goat anti-rabbit and Alexa 555 goat anti-mouse secondary antibodies. Images were taken under deconvolution microscopy and a 'nearest neighbor' analysis was employed as the deconvolution software algorithm (Slidebook, Intelligent Imaging Innovations).