Motoneuron Differentiation of Induced Pluripotent Stem Cells from SOD1G93A Mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder mainly affecting motor neurons. Mutations in superoxide dismutase-1 (SOD-1) account for about 20% of familial ALS patients. A robust supply of motoneurons carrying the mutated gene would help understand the causes of motoneuron death and develop new therapeutics for the disease. Here, we established induced pluripotent stem (iPS) cell lines from SOD1G93A mice and compared their potency in motoneuron generation with normal iPS cells and mouse embryonic stem cells (E14). Our results showed that iPS cells derived from SOD1G93A mice possessed the similar potency in neuronal differentiation to normal iPS cells and E14 cells and can be efficiently driven to motoneuron-like phenotype. These cells exhibited typical neuronal morphology, expressed key motoneuron markers, including ChAT and HB9, and generated repetitive trains of action potentials. Furthermore, these neurons highly expressed human SOD-1 and exhibited shorter neurites compared to controls. The present study provides evidence that ALS-iPS cells can be used as disease models in high-throughput screening and mechanistic studies due to their ability to efficiently differentiate into specific neuronal subtypes.


Introduction
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the selective loss of motoneurons in the cerebral cortex, brainstem, and spinal cord, leading to atrophy of limb, axial, and respiratory muscles [1]. Mutations in superoxide dismutase-1 (SOD-1) account for about 20% of familial ALS patients [2,3]. SOD1G93A mice is a widely accepted model for the ALS research, which express mutant G93A of human SOD-1 and develop clinical symptoms similar to those seen in ALS patients [4]. Motoneurons from SOD1G93A mice could give some information to study the mechanism of ALS [5,6]. A robust supply of motoneurons carrying the genes responsible for this condition would help understand the causes of motoneuron death in ALS and develop new therapeutics for the disease.
Recently, somatic cells can be reprogrammed to a pluripotent state through viral transduction of four transcription factors Oct4, Sox2, c-Myc, and Klf4 [7][8][9]. The induced pluripotent stem (iPS) cells were indistinguishable from ES cells in proliferative and developmental potential, and they can differentiate into derivatives of all germ layers. Several protocols have been developed to induce iPS cells to efficiently differentiate into neurons [10][11][12][13][14]. However, it remains unknown whether iPS cells with genetic deficiency possess neuronal differentiation potential similar to normal cells lines.
In this study, we compared the neuronal differentiation potential between iPS cells derived from SOD1G93A mice and iPS cells derived from normal C57BL/6 mice and investigated whether SOD1 mutations could influence the neuronal differentiation, especially motoneuron generation from iPS cells. Results of the present study would provide evidence on the possibility of the efficient generation of motoneurons from iPS cells with SOD mutations.

Generation and characterization of iPS cells from tail-tip fibroblasts
Totally 6 iPS cell lines were generated by retroviral expression of mouse Oct4, Sox2, c-Myc, and Klf4 from B6SJL-TgN TTFs and C57BL/6 TTFs for characterization and comparison, in which 3 iPS cell lines were derived from 3 transgenic B6SJL-TgN mice (ALS-iPS) and 3 iPS cell line were derived from 3 C57BL/6 mice (C57-iPS) (Figs. 1A and 1C). To confirm that these iPS cells exhibit ES-like properties, we examined some ES cell markers that included alkaline phosphatase (AP) activity and ES cell-specific transcription factors Oct4 and SSEA-1. Results shown in Figs. 1B and 1D demonstrated that the iPS clones exhibited high AP activity. The selected iPS clones were also shown to be positive for Oct4 and SSEA-1 ( Figs. 2A and 2B). To assess the gene expression pattern of the iPS clones, we isolated RNA from iPS cells and the result indicated that the endogenous Oct4, Sox2, c-Myc, Klf4, and Nanog were expressed which confirmed activation of these loci. Results shown in Fig. 2C demonstrated that the transgenes of selected clones from both ALS-iPS-1 and C57-iPS-12 cells were silenced. Importantly, all analyzed iPS clones induced expression from the endogenous Oct4, Sox2, and Nanog loci, and none of these genes were expressed in the original TTF fibroblasts, further supporting of successful reprogramming. Karyotype analyses demonstrated that all analyzed ALS-iPS-1 clones (Fig. 2G) and C57-iPS-12 clones (data not shown) exhibited a normal karyotype.
To confirm the pluripotency of the iPS cells, we injected iPS cells intramuscularly into nude mice. Teratomas formed 4-6 weeks after injection. HE staining of tumor sections from teratomas dissected 5 weeks after injection demonstrated the presence of cell types of all the 3 germ layers (Figs. 2D-F), including gland tissues (endoderm, Fig. 2D), cartilage tissue (mesoderm, Fig. 2E), and neural tube (ectoderm, Fig. 2F).

Transduction and expression of Ta1 a-tubulin/hrGFP (Ta1) during iPS cell neuronal differentiation
To facilitate to monitor the neuronal differentiation potential, we transduced iPS cell lines (ALS-iPS-1 and C57-iPS-12) with lentivector containing Ta1 a-tubulin promoter. E14 cells were used as control. After antibiotic selection, tansduced undifferentiated mouse iPS cells and E14 cells were obtained.
Under the control of the Ta1 a-tubulin promoter, GFP expression appeared at day 2 of the EBs (Fig. 3). After 4 days treatment with RA, the expression of GFP increased over time (Fig. 3). To identify and separate neurons from differentiated E14 and iPS cells, we used a strategy of GFP-based FACS. FACS analysis indicated that 12.462.6% of ALS-iPS-1 differentiated cells was GFP positive, 13

Motoneuron differentiation and characterization of iPS cell lines
Incubation with RA and SHH led to efficient motoneuron generation from iPS and E14 cell lines. Approximately 83.567.9% b-III-tubulin-positive neurons co-expressed ChAT in ALS-iPS-1 cell culture after 5 days treatment with RA and SHH (Fig. 5A4). When stained with Hb9 (a specific postmitotic motoneuron marker), 24.362.7% Tuj1-positive neurons were found to be Hb9-positive ( . Standard whole-cell patch clamp, current-clamp techniques were then used to study the electrical properties of these motor neurons (Fig. 5E). Both ALS-iPS and C57-iPS cells-derived motoneurons generated repetitive trains of action potentials, suggesting that they were functional (Fig. 5F). However, ALS-iPS cells-derived motoneurons exhibited shorter neuritis (50.567.9 mm compared to 95.8612.5 mm for C57-iPS cells-derived motoneurons, P,0.001, student t-test) (Figs. 6A and B). Quantitative real time RT-PCR showed that these ALS-iPS cells-derived motoneurons highly expressed human SOD-1, while C57-iPS cells-derived did not express any (Fig. 6C).

Discussion
Using iPSCs technology, researchers can achieve ES-like cells without the ethical dilemma. The derivation of iPS cells is of such great importance because of the ease and reproducibility of generating them. Direct reprogramming provides, for the first time, a realistic way of generating sufficient numbers of patientspecific pluripotent stem cells. Such cells could be used for regenerative and therapeutic purposes, as demonstrated in mouse models of, for example, sickle cell anemia and Parkinson's disease, respectively [15,16]. In this study, we generated iPS cells from TTFs of SOD1G93A mice by retroviral constructs encoding Oct4, Sox2, c-Myc and Klf4 and demonstrated that they possessed the similar potency in neuronal differentiation to normal iPS cells and mouse ESCs (E14), providing evidence that they can be used as   disease models in high-throughput screening and mechanistic studies.
We used a neuron-specific promoter to monitor the neural differentiation processes of iPS cells. After antibiotic selection, mouse iPS cells and ESCs expressing the Ta1: hGFP with a high degree of purity and stability could be obtained. Our data have shown that it can be used in studies to monitor the differentiation of pluripotent stem cells to early neurons. Modification of derivatives with fluorescent markers allows for the cell separation under a fluorescence-equipped dissecting microscope or by fluorescence activated cell sorting. More than 95% GFP-positive cells were found to be Tuj1-positive neurons across iPS cell lines and one ES cell line, confirming the validity of the Ta1 a-tubulin promoter-based neuronal selection strategy.
The ability to generate iPS cells and efficiently differentiate them into specific neuronal subtypes will provide powerful new tools to study complex neurogenetic disorders. ALS-iPS cellderived motoneurons are an ideal model to study the disease, because they contain the same nuclear genome as of patients, which helps understand the cellular physiology of the disease and development of drug treatment. Using an established protocol, ALS-iPS cells could be driven to motoneuron-like phenotypes efficiently. Our results were supported by the previous findings using SOD1G93A mES cells that the presence of G93A hSOD1 mutation does not affect early neuron differentiation [17]. These cells expressed key motoneuron markers, including ChAT and HB9, indicating the usefulness of these cells in drug screens and basic research. A key limitation of the study is that no phenotypic changes that may correspond to the disease process were described and future work is also needed to functionally characterize these derived cells for basic neuronal properties.

Conclusion
Here, we reported a successful derivation of mouse iPS cells from SOD1G93A mice and these iPS cells can be induced to differentiation to motorneurons with efficiency similar to that of mouse ES cells and wild type mouse iPS cells. Our preliminary study provides a new ALS disease models for high-throughput screening and mechanistic studies.

Cells culture
All experimental procedures were approved by the Institutional Animal Ethical Committee of Sun Yat-sen University and were conducted according to the Guide for the Care and Use of Laboratory Animal of the National Institute of Health (Publication No. 80-23, revised 1996).

Retroviral infection and iPS cell induction
The protocol for mouse iPS was approved by the Institutional Animal Ethical Committee of Sun Yat-sen University.
Retroviral infection was performed as described by others [7,8] with minor modifications. Plat-E cells are purchased from Cell Biolabs (San Diego, CA 92126 USA). Plat-E cells were seeded at 1610 6 cells per well of a 6-well plate. Next day, the cells were transfected with pMXs vectors carrying Oct4, Sox2, c-Myc and Klf4 cDNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 12 hr, the medium was replaced with fresh medium. Virus-containing supernatants were collected from each plate at 72 hr post-transfection, filtered through a 0.45 mm filter before transduction. Equal volumes of the supernatants were mixed and supplemented with 4 mg/ml polybrene. TTFs were seeded at a density of 5610 4 cells per 6-well plate and incubated in the virus/polybrene-containing supernatants for 24 hr. Four days after infection, the cells were further subcultured on irradiated MEFs in ES medium containing LIF. About two weeks after infection, the colonies were mechanically isolated, and propagated under ES conditions.

Cell line characterization
Alkaline phosphatase staining, immunofluorescence microscopy, semi-quantitative RT-PCR for transgene integration, karyotyping, and teratoma formation were carried out to characterize iPS cell lines. Direct Alkaline phosphatase (AP) activity was analyzed with the alkaline phosphatase substrate BCIP/NBT (Sigma) according to the manufacturer's guidelines. The following primary antibodies were used: anti-Oct-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SSEA1 (Chemicon). Total mRNA was isolated using TRIZOL and 1 mg was used to synthesize cDNA using Murine Leukemia Virus reverse transcriptase (Fermentas) and oligo-dT primers (Fermentas) according to the manufacturer's instruction. Then the samples were subjected to amplification with mouse specific primers. b-actin was used as positive control. The PCR products were analyzed by 1.2% agarose gel electrophoresis and visualized by ethidium bromide staining. The detailed information of primers is listed in Table 1. To form teratomas, approximately 2610 6 cells were injected into hind limb muscle of 5-week-old nude mice. After five weeks, teratomas were dissected and fixed in 4% paraformaldehyde. Samples were embedded in paraffin and processed with hematoxylin and eosin staining.
For motoneuron differentiation, SHH and RA were used as described previously with slight modifications [19,20]. After 2 days culture of dissociated E14 or iPS cells in DFK10 medium to form embryoid bodies (EBs), RA (0.1 mM; Sigma) and SHH (200 ng/ ml; R&D Systems) were added to culture for additional 5 days. To facilitate immunostaining analysis, EBs were plated on laminincoated coverslips in DFK10 medium at day 5 and subsequently supplemented with 10 ng/mL BDNF, 10 ng/mL GDNF, 10 ng/ mL CNTF, and 10 ng/mL IGF (R&D Systems) to aid in neuronal survival.
Total RNAs of motoneurons derived from ALS-iPS and C57-iPS cells were extracted using TRIzol (Invitrogen) respectively. Quantitative real time RT-PCR (qPCR) was performed using a Thermal Cycler DiceTM Real Time System and SYBR Premix EX TaqTM (Takara). The primer for human SOD1 (Forward 59-CAT CAG CCC TAA TCC ATC TGA-39 and Reverse 59-CGC GAC TAA CAA TCA AAG TGA-39) was used. b-actin was used for qPCR normalization, and all items were measured in triplicate.

FACS sorting
For flow cytometry analysis, differentiated cells on day 6 were trypsinized and filtered through a 40 mm nylon mesh to remove cell debris. The cells were resuspended in PBS at a concentration of 1610 6 cells/ml. Cell analysis and sorting were performed on a FACS-Aria (BD-Biosciences), through which cell sorting purity of .97% was achieved consistently. An aliquot of untransfected E14 cell suspension was used as negative control. The cells were analyzed by light forward and side scatter by a 488-nm laser beam. Sorting procedures were only based on fluorescence intensity and performed with a flow rate of 1500 events/sec. GFP-positive cells were replated on poly-L-ornithin/laminin-coated dishes in N2B27 medium for 24 hr and were then analyzed for b-III-tubulin expression.

Statistical analysis
Five wells per experiment were imaged for quantification. Results are the average 6 SEM of data from a minimum of three experiments unless stated otherwise. Around 200 cells were counted for each marker. Statistical analysis was performed using one way ANOVA. In case of small numbers in the contingency table, a two-tailed Fisher's-exact test was used.