**3. In vitro models to study ALS**

*In vitro* models are extremely helpful to study human diseases because they allow to analyze different cell types independently from each other and to perform dynamic studies on isolated cells. Moreover, diseased cells can be combined with healthy ones to better understand which cell type is the most critical in the different stages of the disease.

Some of these *in vitro* models were developed using the ALS animal models previously discussed. However, neural cells are impossible to obtain from patients and their extraction

euthanasia is delayed, 3 years. These findings suggest that this disease afflicting dogs is closely related to ALS and identify canine degenerative myelopathy to be the first recognized spontaneously occurring animal model for ALS. The canine ALS model may be particularly valuable for evaluating therapeutic interventions as the environmental

Pigs, although not easily kept for laboratory research, are readily available for biomedical research through the large-scale industrial production of pigs produced for human consumption. Recent research has facilitated the biological experimentation with pigs, and

The domesticated pig (*Sus scrofa*) shares several similarities with human, in particular the size of organs and various aspects of anatomy and physiology. The development of somatic cloning technology and the merger with techniques of targeted genetic modification and conditional gene expression will enhance the possibilities for creating useful models for human diseases in pigs. The pig has also evolved as the major target species for producing xenografts in order to provide appropriate human organs. The sequencing of the domestic pig genome has not yet been fully completed. However, initial draft revealed that the size and composition of the porcine genome is comparable to that of humans; comprising about 2.7 billion base pairs (Hart, et al., 2007). Furthermore, both gene content and sequence are highly conserved between pig and human. Detailed information on the porcine genome together with emerging transgenic technologies, such as siRNA or conditional knockouts will enhance our possibilities to create useful pig models. Other advantages of using domestic pigs to model human diseases include high fertility, great abundance, rapid growth, anatomy and physiology not too different from human and the possibility to introduce genetic modifications in its genome. The pig has been an essential and very successful model in biomedical research and is particularly suited to close the gap between basic research in current models and clinical application. The future will certainly see

The high resemblance between the central nervous systems of humans and pigs makes the pig an ideal model organism for studying human neurodegenerative diseases. For neurodegenerative disorders such as ALS, Parkinson's disease and Alzheimer's disease, the pig may represent a model superior to other models presently available. Large animals, including pigs and non-human primates in neuroscience enable the use of conventional clinical brain imaging and the direct testing of surgical procedures. The evaluation of novel therapeutic avenues in an animal model with higher brain complexity will allow a more

*In vitro* models are extremely helpful to study human diseases because they allow to analyze different cell types independently from each other and to perform dynamic studies on isolated cells. Moreover, diseased cells can be combined with healthy ones to better

Some of these *in vitro* models were developed using the ALS animal models previously discussed. However, neural cells are impossible to obtain from patients and their extraction

understand which cell type is the most critical in the different stages of the disease.

conditions and the SOD1 level of expression mimic better the human ALS situation.

helped develop the pig into a novel model organism for biomedical research.

several promising porcine models for human diseases.

direct translation to human diseases.

**3. In vitro models to study ALS** 

**2.2.5 Pig** 

from post-mortem tissues is limited due to the difficulty to isolate living cells from adult brain or spinal cord, especially motor neurons.

Thus, such postmortem tissue biopsies are mostly used to perform histological and immunohistochemical, genetic as well as proteomic studies. More recently, they were also used to extract neural precursor cells that were further differentiated into motor neurons and glial cells to develop innovative models of the disease.

### **3.1 Organotypic cultures of spinal cord slices**

The best way to preserve all the cellular content and the complex electrophysiological and biochemical organization of cells in the spinal cord is to maintain a whole tissue slice in an organotypic culture. In addition, organotypic slice cultures can be obtained from both embryos and postnatal animals, conferring a major advantage when using transgenic mice from which the disease genotype has to be ascertained after birth (Kosuge, et al., 2009; Mazzone and Nistri, 2011). After dissection of the lumbar spinal cord and removal of the meninges, 200 to 400µm-thick transversal sections are sectioned and transferred into membrane inserts fitting six-well or 12-well culture plates (Caldero, et al., 2010). These organotypic cultures can be used for more than 2 months (Delfs, et al., 1989). Various types of molecules can be added in the culture medium, such as kainate or lithium, to modulate neurotoxicity (Caldero, et al., 2010; Mazzone and Nistri, 2011).

These tissue sections can then be used to perform various analysis like recording the neuron rhythmic activity by placing the tissue on a multielectrode array (Tscherter, et al., 2001; Young, et al., 2007), or studying real-time glutamate release using a biosensor and redox reaction current using a multichannel potentiostat (Mazzone and Nistri, 2011), in addition to the conventional immunohistochemical staining and electrophysiological recordings (Caldero, et al., 2010; Young, et al., 2007). Neonatal brainstem slices can be used to analyze excitotoxicity on hypoglossal motor neurons that control the respiratory drive. These neurons are particularly useful for electrophysiological studies because they exhibit a range of rhythmic patterns that will be altered during early onset of ALS (Cifra, et al., 2011a; Cifra, et al., 2011b).

Organotypic culture is a convenient culture system that closely reproduces the *in vivo* situation and allows dynamic studies with various drugs and useful analysis systems. However, it does not always fully recapitulate what is happening *in vivo* (Tovar, et al., 2009). In addition, it does not easily allow the study of different mixed cell combinations by removing or by adding specific cell types to the tissue slices. These organotypic spinal cord slice cultures are difficult to obtain from postmortem patients due to limited availability and it is obviously impossible to study disease progression using human spinal cord biopsies as opposed to biopsies obtained from animal models.

#### **3.2 Spinal cord cell cultures**

The isolation of individual cells is extremely useful to study various intracellular mechanisms from proliferation to mRNA expression, mitochondrial function, protein aggregation, intermediate filament assembly or axonal transport. However, neurons cannot proliferate and are difficult to maintain in culture. In addition, adult nervous tissues are difficult to access and the viability of the cells extracted from them is very low. Actually, most of the cells that can be maintained in culture after extraction are neural precursor cells (Haidet-Phillips, et al., 2011).

To overcome this limitation, embryos are preferred for nervous tissue harvesting. Indeed, at the E12-13 embryonic developmental stage, the spinal cord of mice embryos is easy to access

*In Vivo* and *In Vitro* Models to Study Amyotrophic Lateral Sclerosis 105

(neurite pattern) closer to adult motor neurons (Bar, 2000). To reproduce an ALS phenotype, motor neurons can be induced to express multiple copies of the gene of interest, for example, mutated human G93A SOD1, by microinjection of the vectors into the cells identified by their specific morphology (Tradewell, et al., 2011; Tradewell and Durham, 2010). Motor neurons can also be purified and cultured separately, to allow their adenoviral transduction with G93A SOD1 (Lunn, et al., 2009), and then, to enhance their survival, they can be plated on a glial cell feeder layer treated with arabinofuranosyl cytidine (AraC), to prevent cell proliferation (De Paola, et al., 2008). These *in vitro* cultures are particularly useful to study cellular morphometry (axon length), gene expression using RT-PCR (ideally with only one purified cell type), apoptosis, signaling pathways, calcium imaging,

The major drawbacks of spinal cord cell cultures are the need to perform cell extraction from embryos for each new experiment. In addition, cells cultured on plastic dishes cannot recapitulate the *in vivo* environment and some results obtained *in vitro* may not be reproducible *in vivo* at least in part because the complex interactions developed in the nervous system are lost. Furthermore, another disadvantage to use primary cells from embryos is that some features of the adult phenotype may not be expressed at this early stage (Park, et al., 2004).

To greatly facilitate *in vitro* studies, motor neuron-enriched embryonic mouse spinal cord cells were fused with mouse neuroblastoma to generate the hybrid cell line NCS-34 (Cashman, et al., 1992; Durham, et al., 1993; Hunter, et al., 1991). This cell line contains small proliferative and undifferentiated cells and larger multinucleate cells. These cells express properties of motor neurons such as choline acetyltransferase, acethylcholine synthesis and neurofilament expression. They respond to agents affecting voltage-gated ion channels, cytoskeletal organization and axonal transport similarly with primary motor neurons, but failed to reproduce synaptic connections (Durham, et al., 1993). This cell line is widely used in several studies related to ALS (Foran, et al., 2011; Tabata, et al., 2008; Vijayalakshmi, et al., 2009; Yang, et al., 2010). For example, NCS-34 cells were treated with the cerebrospinal fluid (CSF) collected from sporadic ALS patients. It induced intracellular aggregates formation, ubiquitin immunoreactivity, neurofilament phosphorylation and choline acetyl transferase expression through induction of endoplasmic reticular stress. This effect was attenuated by addition of VEGF prior to exposition to CSF (Kulshreshtha, et al., 2011; Vijayalakshmi, et al., 2011; Vijayalakshmi, et al., 2009). NCS-34 cells can also be used to screen antioxidant molecules capable of rescuing them from expression of mutated SOD1 (Barber, et al., 2009).

The *Xenopus Laevi* is an African frog from which the oocyte (egg) is a commonly used model for electrophysiological recording because of its large size (about 1mm in diameter) which facilitates its handling. The oocyte has been used for decades to study various ion channels, transporters and receptors. This cell can be transduced with a molecules of interest for coexpression studies including the human glutamate transporter GLT1 (EAAT2) or NMDA receptors (Boehmer, et al., 2006; Texido, et al., 2011; Trotti, et al., 2001). This model has been used in some studies related to ALS, such as the analysis of the properties of the GLT1 transporter activities under controlled voltage clamp in presence or not of mutant SOD1

mitochondrial membrane potential, glutamate uptake, excitotoxicity, etc.

**3.4 Xenopus oocyte model for electrophysiological recordings** 

**3.3 NCS-34 motor neuron cell line** 

(Trotti, et al., 1999).

and remove, and motor neurons, astrocytes and microglia can be extracted, purified and maintain in culture with high viability yields (Gingras, et al., 2007b; Sanelli, et al., 2007; Schnaar and Schaffner, 1981). These cells can be dissociated from the spinal cord and cultured together to facilitate motor neuron survival (Tradewell, et al., 2011; Tradewell and Durham, 2010). Motor neurons will be easy to identify because of their large cell body (>20µm in diameter) and dendritic trees, and can be stained for expression of the transcription factor Hb9 and choline acetyltransferase, specific molecular markers of motor neuron (Fig 3) (Gingras, et al., 2007b; Tradewell, et al., 2011).

Fig. 3. E12 mouse embryo-purified motor neurons.

A: phase contrast microscopy of purified motor neurons 24h after seeding. B: Motor neurons were labeled in red with Hb9 and in green with MAP2. Bar represent 30 μm in A and 50µm in B. *Courtesy of Dr. Marie Gingras.*

Cultures of purified motor neurons without the trophic support of glial cells are difficult to maintain for more than 2 weeks (Bar, 2000; Lunn, et al., 2009). In order to perform studies on a chronic disease like ALS, especially while looking for long-term cell survival effects, dissociated spinal cord cell cultures constitute a better choice as they can be maintain in culture for up to 7 weeks (Tradewell, et al., 2011). Purified motor neurons can also be cultured on an astrocyte feeder layer for several weeks on which they display characteristics

and remove, and motor neurons, astrocytes and microglia can be extracted, purified and maintain in culture with high viability yields (Gingras, et al., 2007b; Sanelli, et al., 2007; Schnaar and Schaffner, 1981). These cells can be dissociated from the spinal cord and cultured together to facilitate motor neuron survival (Tradewell, et al., 2011; Tradewell and Durham, 2010). Motor neurons will be easy to identify because of their large cell body (>20µm in diameter) and dendritic trees, and can be stained for expression of the transcription factor Hb9 and choline acetyltransferase, specific molecular markers of motor

A: phase contrast microscopy of purified motor neurons 24h after seeding. B: Motor neurons were labeled in red with Hb9 and in green with MAP2. Bar represent 30 μm in A and 50µm

Cultures of purified motor neurons without the trophic support of glial cells are difficult to maintain for more than 2 weeks (Bar, 2000; Lunn, et al., 2009). In order to perform studies on a chronic disease like ALS, especially while looking for long-term cell survival effects, dissociated spinal cord cell cultures constitute a better choice as they can be maintain in culture for up to 7 weeks (Tradewell, et al., 2011). Purified motor neurons can also be cultured on an astrocyte feeder layer for several weeks on which they display characteristics

neuron (Fig 3) (Gingras, et al., 2007b; Tradewell, et al., 2011).

Fig. 3. E12 mouse embryo-purified motor neurons.

in B. *Courtesy of Dr. Marie Gingras.*

(neurite pattern) closer to adult motor neurons (Bar, 2000). To reproduce an ALS phenotype, motor neurons can be induced to express multiple copies of the gene of interest, for example, mutated human G93A SOD1, by microinjection of the vectors into the cells identified by their specific morphology (Tradewell, et al., 2011; Tradewell and Durham, 2010). Motor neurons can also be purified and cultured separately, to allow their adenoviral transduction with G93A SOD1 (Lunn, et al., 2009), and then, to enhance their survival, they can be plated on a glial cell feeder layer treated with arabinofuranosyl cytidine (AraC), to prevent cell proliferation (De Paola, et al., 2008). These *in vitro* cultures are particularly useful to study cellular morphometry (axon length), gene expression using RT-PCR (ideally with only one purified cell type), apoptosis, signaling pathways, calcium imaging, mitochondrial membrane potential, glutamate uptake, excitotoxicity, etc.

The major drawbacks of spinal cord cell cultures are the need to perform cell extraction from embryos for each new experiment. In addition, cells cultured on plastic dishes cannot recapitulate the *in vivo* environment and some results obtained *in vitro* may not be reproducible *in vivo* at least in part because the complex interactions developed in the nervous system are lost. Furthermore, another disadvantage to use primary cells from embryos is that some features of the adult phenotype may not be expressed at this early stage (Park, et al., 2004).

### **3.3 NCS-34 motor neuron cell line**

To greatly facilitate *in vitro* studies, motor neuron-enriched embryonic mouse spinal cord cells were fused with mouse neuroblastoma to generate the hybrid cell line NCS-34 (Cashman, et al., 1992; Durham, et al., 1993; Hunter, et al., 1991). This cell line contains small proliferative and undifferentiated cells and larger multinucleate cells. These cells express properties of motor neurons such as choline acetyltransferase, acethylcholine synthesis and neurofilament expression. They respond to agents affecting voltage-gated ion channels, cytoskeletal organization and axonal transport similarly with primary motor neurons, but failed to reproduce synaptic connections (Durham, et al., 1993). This cell line is widely used in several studies related to ALS (Foran, et al., 2011; Tabata, et al., 2008; Vijayalakshmi, et al., 2009; Yang, et al., 2010). For example, NCS-34 cells were treated with the cerebrospinal fluid (CSF) collected from sporadic ALS patients. It induced intracellular aggregates formation, ubiquitin immunoreactivity, neurofilament phosphorylation and choline acetyl transferase expression through induction of endoplasmic reticular stress. This effect was attenuated by addition of VEGF prior to exposition to CSF (Kulshreshtha, et al., 2011; Vijayalakshmi, et al., 2011; Vijayalakshmi, et al., 2009). NCS-34 cells can also be used to screen antioxidant molecules capable of rescuing them from expression of mutated SOD1 (Barber, et al., 2009).

### **3.4 Xenopus oocyte model for electrophysiological recordings**

The *Xenopus Laevi* is an African frog from which the oocyte (egg) is a commonly used model for electrophysiological recording because of its large size (about 1mm in diameter) which facilitates its handling. The oocyte has been used for decades to study various ion channels, transporters and receptors. This cell can be transduced with a molecules of interest for coexpression studies including the human glutamate transporter GLT1 (EAAT2) or NMDA receptors (Boehmer, et al., 2006; Texido, et al., 2011; Trotti, et al., 2001). This model has been used in some studies related to ALS, such as the analysis of the properties of the GLT1 transporter activities under controlled voltage clamp in presence or not of mutant SOD1 (Trotti, et al., 1999).

*In Vivo* and *In Vitro* Models to Study Amyotrophic Lateral Sclerosis 107

2007). It was shown that G93A-SOD1 ESCs differentiated into motor neurons had a lower survival over 2 to 4 weeks in culture than their WT counterparts and showed ALS pathology hallmarks such as SOD1 protein aggregates and increase in activated caspase-3 expression after 21 days of culture (Di Giorgio, et al., 2007). Motor neurons have also been successfully differentiated from human ESCs (Hester, et al., 2011; Hu and Zhang, 2009). However, to obtain a source of multipotent stem cells from adult ALS patients, other approaches needed to be developed. These cells can be obtained from easily accessible tissues such as skin (Gingras, et al., 2007a; Toma, et al., 2001), but their limited proliferation capacity *in vitro* needs a relatively large amount of starting materials that up to now preclude their extraction from patients. Another possibility is to obtain postmortem biopsies of brain and spinal cord from ALS patients. It has been shown that if living glial cells and neurons are difficult to isolate from these tissues, neural precursor cells (NPCs) with a good proliferation rate are oppositely easy to purify and culture *in vitro*. These cells can then be differentiated in the cell type of interest. Neurons, astrocytes and oligodendrocytes were differentiated from NPCs purified from familial and sporadic ALS patient postmortem spinal cord samples (Fig 4) to study the toxic properties of patients-derived astrocytes on motor neurons (Haidet-Phillips, et al., 2011). Both FALS and SALS-derived astrocytes were shown to be toxic to motor neurons, and the knock-down of SOD1 in SALS-derived astrocytes was demonstrated to attenuate this toxicity (Haidet-Phillips, et al., 2011). But postmortem tissues are difficult to

A much more versatile technique to generate multipotent stem cells from adults is to generate induced pluripotent stem cells (iPS cells) starting from somatic cells. This technique only requires a small punch biopsy of a few millimeters in diameter collected from the patient's skin, which will heal spontaneously without scarring. From this biopsy, dermal fibroblasts will be extracted and expanded to generate iPS cells. Generation of IPs cells is possible via overexpression of a defined set of transcription factors (*c-Myc*, *Oct3/4*, *Klf4* and *SOX2*) (Takahashi, et al., 2007a; Takahashi, et al., 2007b). The genetic transduction of these oncogenes is usually achieved by using retroviral or lentiviral vectors. However, the use of integrating viral vectors represent a major obstacle to the therapeutic translation of iPS cells as this technology can produce insertional mutagenic lesions that are potentially tumorigenic. Other methods to reprogram cells are now being intensively tested, such as the use of secreted recombinant reprogramming factors present in the culture media. These iPS cells are morphologically and phenotypically similar to embryonic stem (ES) cells and thus offer exciting possibilities in stem cell research and regenerative medicine. This method has been successfully applied to ALS patients and allowed to study iPS-derived motor neurons *in vitro* (Dimos, et al., 2008; Mitne-Neto, et al., 2011). Since this technology is still recent, it is not clear whether motor neurons and glial cells derived from ALS patients-iPS cells will efficiently recapitulate the disease *in vitro*. However, these cells seem to behave like ESCs and even if they only partly mimic the ALS phenotype in culture, they certainly will be

extremely interesting to use, especially for a better understanding of sporadic ALS.

Now that iPS or NPCs ALS patient-derived motor neurons and glial cells will be available to develop better ALS *in vitro* models, their design could also be improved. The culture of neural cells in two dimensions on plastic does not mimic properly the *in vivo* situation. Building three-dimensional environment around ALS patient-derived neural cells could be a

**3.6 Three-dimensional in vitro models** 

obtain.

### **3.5 Neural precursor cells (NPCs) and induced pluripotent stem cells (iPS)**

One important advantage of *in vitro* cell cultures could gain over animal models would be to collect neural precursor cells directly from patients. These cells can be differentiated into all neural cell types, including motor neurons. As previously discussed, most of animal and cell culture models developed to study ALS so far were derived from known FALS-associated mutations in the *SOD1*, *TARDBP* or *FUS/TLS* genes. While these models are particularly useful to study FALS, they are not ideal for the study of SALS. Therefore, trying to understand the cause of the disease through the study of motor neurons and glial cells obtained from SALS patients would be challenging but potentially highly promising. However, motor neurons, and even glial cells are extremely difficult to isolate from brain and spinal cord samples obtained from post-mortem ALS patients.

Fig. 4. Characterization of neural precursor cells (NPCs) extracted from postmortem ALS patient's spinal cord.

Cells express Iba1 (a marker of microglia) and 3-tubulin (early marker of neuronal differentiation) (A), Vimentin (transiently expressed in neuronal precursors) (B), Nestin (transiently expressed in neuronal precursors) (C) and Islet-1 (an early marker for motor neuron differentiation) (C) that highlight their NPC status when co-expressed together in the same cells. *Courtesy of Dr. Marie-Josée Beaulieu.*

One very promising alternative is to take advantage of the neural precursor cells that can be extracted from adult tissues. Indeed, in a first step, the potential of mouse embryonic stem cells (ESCs) to differentiate into motor neurons has been well established (Wichterle, et al., 2002) and applied to the development of *in vitro* ALS models using ALS-linked G93A *SOD1* gene mutation and WT SOD1 mouse embryos as source of stem cells (Di Giorgio, et al.,

One important advantage of *in vitro* cell cultures could gain over animal models would be to collect neural precursor cells directly from patients. These cells can be differentiated into all neural cell types, including motor neurons. As previously discussed, most of animal and cell culture models developed to study ALS so far were derived from known FALS-associated mutations in the *SOD1*, *TARDBP* or *FUS/TLS* genes. While these models are particularly useful to study FALS, they are not ideal for the study of SALS. Therefore, trying to understand the cause of the disease through the study of motor neurons and glial cells obtained from SALS patients would be challenging but potentially highly promising. However, motor neurons, and even glial cells are extremely difficult to isolate from brain

Fig. 4. Characterization of neural precursor cells (NPCs) extracted from postmortem ALS

One very promising alternative is to take advantage of the neural precursor cells that can be extracted from adult tissues. Indeed, in a first step, the potential of mouse embryonic stem cells (ESCs) to differentiate into motor neurons has been well established (Wichterle, et al., 2002) and applied to the development of *in vitro* ALS models using ALS-linked G93A *SOD1* gene mutation and WT SOD1 mouse embryos as source of stem cells (Di Giorgio, et al.,

Cells express Iba1 (a marker of microglia) and 3-tubulin (early marker of neuronal differentiation) (A), Vimentin (transiently expressed in neuronal precursors) (B), Nestin (transiently expressed in neuronal precursors) (C) and Islet-1 (an early marker for motor neuron differentiation) (C) that highlight their NPC status when co-expressed together in

patient's spinal cord.

the same cells. *Courtesy of Dr. Marie-Josée Beaulieu.*

**3.5 Neural precursor cells (NPCs) and induced pluripotent stem cells (iPS)** 

and spinal cord samples obtained from post-mortem ALS patients.

2007). It was shown that G93A-SOD1 ESCs differentiated into motor neurons had a lower survival over 2 to 4 weeks in culture than their WT counterparts and showed ALS pathology hallmarks such as SOD1 protein aggregates and increase in activated caspase-3 expression after 21 days of culture (Di Giorgio, et al., 2007). Motor neurons have also been successfully differentiated from human ESCs (Hester, et al., 2011; Hu and Zhang, 2009). However, to obtain a source of multipotent stem cells from adult ALS patients, other approaches needed to be developed. These cells can be obtained from easily accessible tissues such as skin (Gingras, et al., 2007a; Toma, et al., 2001), but their limited proliferation capacity *in vitro* needs a relatively large amount of starting materials that up to now preclude their extraction from patients. Another possibility is to obtain postmortem biopsies of brain and spinal cord from ALS patients. It has been shown that if living glial cells and neurons are difficult to isolate from these tissues, neural precursor cells (NPCs) with a good proliferation rate are oppositely easy to purify and culture *in vitro*. These cells can then be differentiated in the cell type of interest. Neurons, astrocytes and oligodendrocytes were differentiated from NPCs purified from familial and sporadic ALS patient postmortem spinal cord samples (Fig 4) to study the toxic properties of patients-derived astrocytes on motor neurons (Haidet-Phillips, et al., 2011). Both FALS and SALS-derived astrocytes were shown to be toxic to motor neurons, and the knock-down of SOD1 in SALS-derived astrocytes was demonstrated to attenuate this toxicity (Haidet-Phillips, et al., 2011). But postmortem tissues are difficult to obtain.

A much more versatile technique to generate multipotent stem cells from adults is to generate induced pluripotent stem cells (iPS cells) starting from somatic cells. This technique only requires a small punch biopsy of a few millimeters in diameter collected from the patient's skin, which will heal spontaneously without scarring. From this biopsy, dermal fibroblasts will be extracted and expanded to generate iPS cells. Generation of IPs cells is possible via overexpression of a defined set of transcription factors (*c-Myc*, *Oct3/4*, *Klf4* and *SOX2*) (Takahashi, et al., 2007a; Takahashi, et al., 2007b). The genetic transduction of these oncogenes is usually achieved by using retroviral or lentiviral vectors. However, the use of integrating viral vectors represent a major obstacle to the therapeutic translation of iPS cells as this technology can produce insertional mutagenic lesions that are potentially tumorigenic. Other methods to reprogram cells are now being intensively tested, such as the use of secreted recombinant reprogramming factors present in the culture media. These iPS cells are morphologically and phenotypically similar to embryonic stem (ES) cells and thus offer exciting possibilities in stem cell research and regenerative medicine. This method has been successfully applied to ALS patients and allowed to study iPS-derived motor neurons *in vitro* (Dimos, et al., 2008; Mitne-Neto, et al., 2011). Since this technology is still recent, it is not clear whether motor neurons and glial cells derived from ALS patients-iPS cells will efficiently recapitulate the disease *in vitro*. However, these cells seem to behave like ESCs and even if they only partly mimic the ALS phenotype in culture, they certainly will be extremely interesting to use, especially for a better understanding of sporadic ALS.

#### **3.6 Three-dimensional in vitro models**

Now that iPS or NPCs ALS patient-derived motor neurons and glial cells will be available to develop better ALS *in vitro* models, their design could also be improved. The culture of neural cells in two dimensions on plastic does not mimic properly the *in vivo* situation. Building three-dimensional environment around ALS patient-derived neural cells could be a

*In Vivo* and *In Vitro* Models to Study Amyotrophic Lateral Sclerosis 109

In A, after the culture of fibroblasts and Schwann cells for 21 days in a collagen sponge (blue area), mouse motor neurons were seeded on top of the sponge (purple area) and cultured for an additional 14 days and observed by histology staining with Masson's trichrom. In B, axonal migration (stained in green with an antibody against Neurofilament-M) originating from the top layer was shown migrating through the connective tissue. In C, a transmission electron microscopic picture showed that some neurites were wrapped with thick myelin sheaths, observed at higher magnification in D. Bar in A, 60µm; bar in B, 100µm; bar in C,

A very large body of knowledge has been built over the years with the development of various *in vivo* models to better understand ALS. Particularly, the generation of a large number of different transgenic mice has been extremely useful to study familial ALS, but has met with limited success so far to study SALS or to identify drugs alleviating the disease symptoms. This could be explained by the existence of redundant compensatory mechanisms present in higher vertebrates. The use of invertebrates together with higher animal models will hopefully allow the identification of novel pathogenic pathways and

In parallel, the development of *in vitro* models will give valuable information about the intracellular modulations induced by the disease. In addition, the promise of building sophisticated *in vitro* models using patient's cells will also be crucial to better understand the disease. However, the results obtained from *in vitro* models may not always be relevant because they are oversimplified compared to the *in vivo* situation. Therefore, the best strategy to reduce the limitations inherent to all these different models, and to benefit from their specific advantages is to combine both *in vivo* and *in vitro* models in order to better model the disease. Future work, focusing on different neuronal in vitro system and animal models, will certainly increases our knowledge in the biology of ALS and hopefully leads to more translational researches in order to find a cure or to slow down the course of this yet

We would like to thank Dr. Alex J. Parker and Dr Guy A. Rouleau, from the Centre of Excellence in Neuromics, University of Montreal, Quebec, Canada, for having provided respectively the figure 1 and 2 of this book chapter. We also thank Marie Gingras and Marie-Josée Beaudet for their assistance in generating figure 3 to 5. Dr François Gros-Louis is supported by the Fond de la recherche en santé du Québec (FRSQ) and by the Fondation des hôpitaux Enfant-JésusSt-Sacrement. Dr François Berthod is supported by the Muscular Dystrophy Association USA (www.mda.org). The authors declare no conflict of interest.

Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G.,

Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., George, R. A., Lewis, S. E., Richards, S., Ashburner, M., Henderson, S. N., Sutton, G. G., Wortman, J. R., Yandell, M. D., Zhang, Q., Chen, L. X., Brandon, R. C., Rogers, Y. H., Blazej, R. G., Champe,

2µm; bar in F, 0,2µm *(Modified from Gingras, et al., 2008)*.

novel therapeutic avenues in order to cure this devastating disease.

**4. Conclusion** 

untreatable disease.

**6. References** 

**5. Acknowledgment** 

major improvement in the development of *in vitro* models of the disease. The culture of NPCs in methylcellulose scaffold enriched with laminin has been shown to reduce apoptosis and enhance survival, differentiation into neurons, astrocytes or oligodendrocytes and neurite extension (Cullen, et al., 2007; Stabenfeldt, et al., 2010). Motor neurons axonal migration can also be analyzed when these cells are cultured on the top of a threedimensional reconstructed connective tissue made of a collagen sponge populated with fibroblasts. The addition of neurotrophic factors underneath the construct promotes neurite growth from the top to the bottom of the tissue. The main advantage of this model is to enable myelin sheath formation by Schwann cells around axons in the connective tissue and the possibility to add various glial cells (astrocytes, microglia) in the motor neuron layer (Fig 5) (Gingras, et al., 2008). In addition, it is possible to easily combine cells from different origin, like WT motor neurons with ALS glial cells, and vice-versa in order to further explore the non-cell autonomous effect observed in SOD1-linked ALS pathology. Such combinations could also be very informative in the study of sporadic ALS. Alternatively, a 3 dimensional reconstructed muscle model has been developed to study *in vitro* the effect of muscle stretching on mRNA expression of muscle cells. This model was prepared using primary muscle cultures from human control subjects and ALS patients. The cells were embedded in a collagen gel tethered to a Culture Force Monitor to analyze gel contraction (Cheema, et al., 2003; Evans, et al., 2010).

Finally, the combination of ALS patient NPC-derived neural cells in tissue-engineered reconstructed spinal cord models is a promising strategy to develop the next generation of *in vitro* models of ALS.

Fig. 5. Three-dimensional model of motor neuron axonal migration and myelin sheath formation.

In A, after the culture of fibroblasts and Schwann cells for 21 days in a collagen sponge (blue area), mouse motor neurons were seeded on top of the sponge (purple area) and cultured for an additional 14 days and observed by histology staining with Masson's trichrom. In B, axonal migration (stained in green with an antibody against Neurofilament-M) originating from the top layer was shown migrating through the connective tissue. In C, a transmission electron microscopic picture showed that some neurites were wrapped with thick myelin sheaths, observed at higher magnification in D. Bar in A, 60µm; bar in B, 100µm; bar in C, 2µm; bar in F, 0,2µm *(Modified from Gingras, et al., 2008)*.
