**3. Stem cell therapy for ALS**

### **3.1 The motor neuron replacement strategy**

As motor neuron loss is the key diagnostic feature of ALS, the most straightforward strategy is to derive motor neurons from various types of stem cells and try to use them to replace the dead motor neurons in patients. For adult stem cells, cells expressing neuron and glial lineage markers were successfully derived from trans-differentiation of human umbilical cord blood cells (McGuckin et al., 2004) and mouse bone marrow stem cells (Croft et al. 2006). However, those cells' electrophysiological properties, survival, differentiation, and efficacy of integration to functional neurons and glial cells either *in vitro* or *in vivo* were not tested. Neural stem cells are the only type of adult stem cells which have successfully derived motor neurons that are functional *in vivo* (Gao et al., 2005). Human neural stem cells, which are scarce in the human body, are usually derived from embryonic stem cells or fetal brain tissues (Tai & Svendsen, 2004).

More promising results were shown in experiments using pluripotent stem cells. From mouse embryonic stem (ES) cells, motor neurons were successfully generated by induction of developmentally relevant signaling factors. The derived cells survive when transplanted into chick embryonic spinal cord, extend axons, and exhibit signs of presynaptic specialization when reaching targeted muscles (Wichterle et al., 2002). Another study shows that those cells possess immunohistochemical and electrophysiological features of normal motor neurons (Miles et al., 2004). Similar to mouse ES cells, human ES cells have been reported to form functional neurons (Li et al., 2005; Lee et al., 2007).

Functional motor neurons can also be derived from human induced pluripotent stem (iPS) cells, a possible alternative that may avoid the ethical concerns for the use of human ES cells (Karumbayaram et al., 2009). iPS cells are somatic cells that are reprogrammed into pluripotent stem cells (Yu et al., 2007; Takahashi, 2007), with great similarity to embryonic stem cells. They are capable of deriving patient-specific differentiated cells like neurons and glia, which allows them to potentially be used for autologous cell replacement in ALS patients. iPS cells have been generated from ALS patients and the cells are capable of differentiating into motor neurons (Dimos, 2008). However, introduction of new genes during the production of iPS cells may give rise to additional technical concerns when translating to clinical studies.

Mouse ES-derived motor neurons reportedly grow around the ventral horn when transplanted into the spinal cord of rats with impaired motor neurons (Harper et al., 2004). In combination with chemicals that overcome myelin-mediated repulsion and GDNF that stimulates axon guidance towards skeletal muscles, further improvement in survival and engraftment of the transplanted cells was observed. Improvement in motor function of the paralyzed rats was also observed (Despande et al., 2006).

Despite the excitement that these transplantation studies brings to the field, the fact that these studies were performed on static models of motor neuron loss does not guarantee success in progressive motor neuron diseases like ALS. In addition, in order for the motor neuron replacement strategy to be successful, the transplanted motor neuron will first need to receive synaptic input from the presynaptic neurons and extend it's axon all the way to the targeted muscle at a rate of 1-3 mm/day, which takes months to years in humans, before innervation to the targeted muscle can be possible (Papadeas & Maragakis, 2009). Therefore motor neuron replacement may not be a legitimate treatment at this moment.

### **3.2 The neuroprotection strategy**

440 Amyotrophic Lateral Sclerosis

investigation several interesting findings have been observed and may provide insights to better understand the underlying mechanism of the disease. For example, finding growth factors' targets may help us find how the disease is initiated. Currently, the growth factors' targets are not fully known. It could be the degenerating motor neuron itself, the neighboring neuron, or surrounding glial cells. But a recent report about wild type nonneuronal cells extending survival of SOD1 mutant motor neurons in chimeric ALS mice (Clement et al., 2003) may provide adequate evidence showing that the growth factor's

Another point of interest is the similarity of the growth factors that have been used. All GDNF, IGF-1, VEGF, and BDNF interact with receptor tyrosine kinases to produce downstream effects. Experiments have shown that those growth factors indeed work in a similar pathway and mechanism as there is no additional improvement observed when they work in combination (IGF-1 and VEGF) as compared to working individually (Dodge et al. 2010). Another article reports that VEGF promotes motor neuron survival by blocking Caspase through Phosphoinositide 3-kinase/ protein kinase B (PI3K/Akt) pathway (Lunn et al., 2009). Further investigation on the PI3K/Akt pathway may provide clues on how motor

As motor neuron loss is the key diagnostic feature of ALS, the most straightforward strategy is to derive motor neurons from various types of stem cells and try to use them to replace the dead motor neurons in patients. For adult stem cells, cells expressing neuron and glial lineage markers were successfully derived from trans-differentiation of human umbilical cord blood cells (McGuckin et al., 2004) and mouse bone marrow stem cells (Croft et al. 2006). However, those cells' electrophysiological properties, survival, differentiation, and efficacy of integration to functional neurons and glial cells either *in vitro* or *in vivo* were not tested. Neural stem cells are the only type of adult stem cells which have successfully derived motor neurons that are functional *in vivo* (Gao et al., 2005). Human neural stem cells, which are scarce in the human body, are usually derived from embryonic stem cells or

More promising results were shown in experiments using pluripotent stem cells. From mouse embryonic stem (ES) cells, motor neurons were successfully generated by induction of developmentally relevant signaling factors. The derived cells survive when transplanted into chick embryonic spinal cord, extend axons, and exhibit signs of presynaptic specialization when reaching targeted muscles (Wichterle et al., 2002). Another study shows that those cells possess immunohistochemical and electrophysiological features of normal motor neurons (Miles et al., 2004). Similar to mouse ES cells, human ES cells have been

Functional motor neurons can also be derived from human induced pluripotent stem (iPS) cells, a possible alternative that may avoid the ethical concerns for the use of human ES cells (Karumbayaram et al., 2009). iPS cells are somatic cells that are reprogrammed into pluripotent stem cells (Yu et al., 2007; Takahashi, 2007), with great similarity to embryonic stem cells. They are capable of deriving patient-specific differentiated cells like neurons and glia, which allows them to potentially be used for autologous cell replacement in ALS patients. iPS cells have been generated from ALS patients and the cells are capable of differentiating into motor neurons

reported to form functional neurons (Li et al., 2005; Lee et al., 2007).

target is the supporting glia instead of neurons.

neuron death is triggered in ALS.

**3. Stem cell therapy for ALS** 

**3.1 The motor neuron replacement strategy** 

fetal brain tissues (Tai & Svendsen, 2004).

### **3.2.1 Non-cell autonomous nature of motor neuron death in ALS**

Previously, little attention has been paid to the function of glial cells in the nervous system. However, we now know that glial cells modulate neuronal functions such as glutamate uptake, synaptic plasticity, trophic factor support, and even neuronal transmission (Kirchhoff et al., 2001). Studies also show that motor neuron death in ALS is non-cell autonomous, or mediated by astrocytes and microglia (Hall et al., 1998; Barbeito et al., 2004). Researchers also hypothesize that astrocytes and/or microglia form a positive feedback loop with motor neurons that leads to further propagation of the disease (Rao & Weiss, 2004). Moreover, chimeric mice with increased proportion of healthy, wild type glial cells increase survival of nearby human SOD1 mutant neurons *in vivo* (Clement et al., 2003). Using a CRE-lox system, selective reduction of the mutant gene in microglia and astrocytes in SOD1 transgenic mice slows disease progression, but has no effect on disease onset (Boillée et al., 2006b; Yamanaka et al., 2008).

Additional evidence is provided by stem cell–derived motor neurons/astrocytes co-culture. A study in 2007 shows that primary and ES cell-derived motor neurons are complementary in an *in vitro* motor neuron/astrocytes study for ALS (Nagai et al., 2007). From then on, studies using the following combinations have been performed: hES cell derived motor neurons with primary hSOD1-G93A or wild type mouse primary astrocytes (Di Giorgio et al. 2008); hSOD1- G93A mouse ES derived motor neuron with hSOD1-G93A derived mouse primary astrocytes (Di Giorgio 2007); and hES cells derived motor neuron with primary human astrocytes transfected with hSOD1-G47R genes (Marchetto, 2008). The Marchetto paper also uses that approach to verify a potential drug that has been beneficial in ALS rodent models. The success in this approach provides an easily accessible *in vitro* testing platform for cell-cell interactions in ALS and underlying disease mechanisms. Drug discovery will also accelerate as high throughput drug screening can be performed on the cultures.

### **3.2.2 Astrocyte replacement**

Based on non-cell autonomous nature of motor neuron death in ALS, astrocyte replacement is another feasible strategy for ALS stem cell therapy. Researchers transplant

Stem Cell Application for Amyotrophic Lateral Sclerosis: Growth Factor Delivery and Cell Therapy 443

**Cell type Subject Injection Site Effect Paper** 

cells survive and differentiated into mature astrocytes; reduces microgliosis; prolongs survival, ameliorates motor neuron loss and slows down motor function decline; normal GLT-1

Lepore et al.

Beers et al. 2006

Kerr et al. 2003

Garbuzova-Davis et al. 2002

Hemendiner et

al. 2005

2007

No effect Habisch et al.

2008

level

cells effectively differentiated into microglia cells; prolongs survival; suppressed

cytotoxicity; restore glial activation

cells distributed extensively over the rostrocaudal length of the spinal cord and migrated into the spinal cord parenchymal partially recovered motor function 12 and

24 weeks after transplantation

motor neuron survival; no effect on disease onset and progression

survival,

delay onset, prolong

significant increase in

life span Corti et al. 2004

bilateral cervival spinal cord injection

i.p. injection

i.c.v injection

L4-L5 segments of the ventral horn spinal cord

unilateral spinal injection into the L4-L5 ventral horn

direct injection into the CSF (the cisterna magna).

(CSF)

G93A mice i.p. injection delayed onset, increase

Mouse GRP hSOD1-

Mouse bone marrow cell

Mouse Bone marrow transplant

Human embryonic germ cell

hNT cell hSOD1-

Mouse Sertoli

Neuroectoderm al derivatives of hUBS (hUBS-NSCs)

cell

G93A rats

hSOD1- G93A /PU1-/ double mutant mice

hSOD1-

rats with diffused motor neuron injury

G93A mice

hSOD1- G93A

hSOD1- G93A

glial restricted precursor (GRP) cells (lineage-restricted as derived from developing spinal cord) focally to cervical spinal cord that controls respiratory function in SOD1 rats (Lepore et al., 2008). The effect of the GRP transplant is significant: GRP cells survive and differentiated into mature astrocytes *in vivo.* The treatment also reduces microgliosis, prolongs survival, ameliorates motor neuron loss, and slows motor function decline. The group also found that the ALS rats with grafted GRP cells maintain normal level of glutamate transporter (GLT-1), an astrocyte-specific protein that has reduced expression in both ALS model rats and human patients (Howland et al., 2002; Rothstein et al., 1995). This may provide further evidence that astrocyte replacement is a sound strategy for ALS cell therapy.

### **3.2.3 Immunomodulation**

Other than replacement strategies, some stem cell therapies modulate the immunological environment around the degenerating motor neurons to prevent them from dying. Bone marrow cells provide a rich source of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). HSCs can give rise to a great variety of blood cells and cells in the immune system, but will particularly differentiate into microglia when introduced to the nervous system (Vitry et al., 2003). MSCs do not have the ability to differentiate into cells in the nervous system, but contribute to improved locomotion by differentiating into cells in the skeletal muscle lineage (Corti et al., 2004). Bone marrow transplanted into irradiated SOD1G93A/PU1-/- double mutants (born without microglia and peripheral immune cells) prolonged survival and slowed disease progression (Beers et al., 2006). Another similar experiment confirms the result (Corti et al., 2004). This led to clinical trials of MSC and HSC transplants to sporadic ALS patients (Appel et al., 2008; Mazzini et al., 2008). Some of these studies show promising results **(Table 1)**.

### **3.3 Protective effect of neural stem cell and other cells in the neural lineage**

Although most transplantations involving cells in the neural lineage were aimed at replacement of motor neurons, researchers now find that neuro-protection was instead the main effect. Various cell transplantations have been performed on hSOD1-G93A rodent models. They include: i) human embryonic germ cell delivered to cerebral spinal fluid (Kerr et al., 2003); ii) human neural stem cells grafted into the spinal cord (Yan et al., 2006); iii) hNT neurons derived from a human teratocarcinoma cell line grafted into spinal cord (Garbuzova-Davis et al., 2002); mouse Sertoli cells into parenchyma (Hemendinger et al., 2005); and human umbilical cord blood cells transfused into the systemic circulation (Habisch et al., 2007). In each of the cases, there was some degree of positive effect on motor neuron survival and life span of the animals. In addition, in most cases the positive effect is related to growth factor release (Suzuki & Svendsen, 2008). However, these studies do not specify which cell types are eventually exerting the protective effect or releasing the growth factors, though they are expected to be astrocytes (See Section 3.2 of this chapter). However, one human neural stem cell (NSC) transplant study suggests that the neuroprotective effect of host motor neurons stems from the ability of NSCs to differentiate into neuronal subtypes other than motor neurons such as GABAergic neurons that forms synaptic connection between grafted and host motor neurons (Xu et al., 2009). These neurons may provide additional benefits other than that from glial cells.

glial restricted precursor (GRP) cells (lineage-restricted as derived from developing spinal cord) focally to cervical spinal cord that controls respiratory function in SOD1 rats (Lepore et al., 2008). The effect of the GRP transplant is significant: GRP cells survive and differentiated into mature astrocytes *in vivo.* The treatment also reduces microgliosis, prolongs survival, ameliorates motor neuron loss, and slows motor function decline. The group also found that the ALS rats with grafted GRP cells maintain normal level of glutamate transporter (GLT-1), an astrocyte-specific protein that has reduced expression in both ALS model rats and human patients (Howland et al., 2002; Rothstein et al., 1995). This may provide further evidence that astrocyte replacement is a sound strategy for ALS

Other than replacement strategies, some stem cell therapies modulate the immunological environment around the degenerating motor neurons to prevent them from dying. Bone marrow cells provide a rich source of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). HSCs can give rise to a great variety of blood cells and cells in the immune system, but will particularly differentiate into microglia when introduced to the nervous system (Vitry et al., 2003). MSCs do not have the ability to differentiate into cells in the nervous system, but contribute to improved locomotion by differentiating into cells in the skeletal muscle lineage (Corti et al., 2004). Bone marrow transplanted into irradiated SOD1G93A/PU1-/- double mutants (born without microglia and peripheral immune cells) prolonged survival and slowed disease progression (Beers et al., 2006). Another similar experiment confirms the result (Corti et al., 2004). This led to clinical trials of MSC and HSC transplants to sporadic ALS patients (Appel et al., 2008; Mazzini et al., 2008). Some of these

**3.3 Protective effect of neural stem cell and other cells in the neural lineage** 

Although most transplantations involving cells in the neural lineage were aimed at replacement of motor neurons, researchers now find that neuro-protection was instead the main effect. Various cell transplantations have been performed on hSOD1-G93A rodent models. They include: i) human embryonic germ cell delivered to cerebral spinal fluid (Kerr et al., 2003); ii) human neural stem cells grafted into the spinal cord (Yan et al., 2006); iii) hNT neurons derived from a human teratocarcinoma cell line grafted into spinal cord (Garbuzova-Davis et al., 2002); mouse Sertoli cells into parenchyma (Hemendinger et al., 2005); and human umbilical cord blood cells transfused into the systemic circulation (Habisch et al., 2007). In each of the cases, there was some degree of positive effect on motor neuron survival and life span of the animals. In addition, in most cases the positive effect is related to growth factor release (Suzuki & Svendsen, 2008). However, these studies do not specify which cell types are eventually exerting the protective effect or releasing the growth factors, though they are expected to be astrocytes (See Section 3.2 of this chapter). However, one human neural stem cell (NSC) transplant study suggests that the neuroprotective effect of host motor neurons stems from the ability of NSCs to differentiate into neuronal subtypes other than motor neurons such as GABAergic neurons that forms synaptic connection between grafted and host motor neurons (Xu et al., 2009). These neurons may provide

cell therapy.

**3.2.3 Immunomodulation** 

studies show promising results **(Table 1)**.

additional benefits other than that from glial cells.


Stem Cell Application for Amyotrophic Lateral Sclerosis: Growth Factor Delivery and Cell Therapy 445

We have introduced two successful strategies for slowing ALS disease progression in the previous sections of this chapter. Although both of them in some degree involve the release of neuroprotective growth factors, both strategies have their shortcomings. In viral delivery of growth factors, the cells still carry the mutant SOD1 gene or has the disease phenotype. Therefore the cells that are delivering the treatment are indeed still doing harm on the surrounding cells at the same time. On the other hand, neuroprotective strategy of stem cell transplants, though increases the proportion of wild type (normal) cells around the injection site(s), the transplanted cells may not naturally produce the desired neuroprotective growth factors in a pharmaceutically adequate amount (Gonzalez, 2009). Therefore, it is reasonable for us to combine the two strategies and see if they can complement each other and produce

Based on the logic above, our group genetically engineered human neural progenitor cells (hNPC) that express and secrete GDNF through lentiviral infection (Klein et al., 2005; Suzuki et al., 2007). hNPC are comprised of multiple classes of neural stem cells and lineagerestricted precursors. They are isolated from fetal brain cortical tissue (Svendsen et al., 1996; Keyoung et al., 2001; Tamaki et al., 2006; Suslov, 2002) and can be maintained for over 50 weeks in the presence of mitogen while retaining the ability to differentiate into astrocytes (Wright et al, 2003). With their special properties, hNPC can thus serve as "mini-pumps" to provide glial replacement and deliver trophic factors through transplantation into specific sites in the brain and spinal cord of diseased animals and patients. hNPC-GDNF were transplanted to the lumbar region of the spinal cord of hSOD1-G93A rats. We observed robust migration of the transplanted cells into the degenerating region, efficient delivery of GDNF, as well as preservation of a large proportion of motor neurons at both early and late stages of the disease within chimeric regions (Suzuki et al. 2007). However, the preservation of motor neurons does not accompany with continued innervations of motor neuron to the

skeletal muscle end plates, thus had no effect on ipsilateral hind limb function.

Skeletal muscles clearly play an important role in guiding and attracting the developing neurons; and provide trophic support to maintain motor neuron function (Dobrowolny et al., 2005). A previous study showed that transplants of genetically engineered myoblasts (a kind of skeletal muscle precursor which has the ability to fuse with mature myofibers) secreting GDNF ameliorates motor neuron loss in ALS mice (Mohajeri et al., 1999). Thus we genetically engineered human MSCs (hMSCs) that express and secrete GDNF and transplanted them to three muscle groups in hSOD1-G93A rats (Suzuki et al., 2008). MSCs can be easily obtained from bone marrow from donations and have the ability to differentiate into the skeletal muscle lineage (Caplan & Arnold, 2009). The transplanted cells survives in the host skeletal muscle and releases GDNF. Moreover, it significantly increases the number of functional neuromuscular junctions and improves motor neuron survival in spinal cord at the mid-stage of disease. Furthermore, intramuscular hMSC-GDNF transplantation remarkably prolongs disease progression, increasing overall life span up to 28 days, which is one of the greatest

**4. Working in combination: Genetically engineered stem cells as a tool of** 

**growth factor delivery for ALS** 

**4.1 hNPC-GDNF injection to spinal cord** 

**4.2 hMSC-GDNF injection to skeletal muscles** 

improvements ever observed in familial ALS model rats.

a great synergic effect.


Table 1. Stem Cell Trials for ALS GRP. Glial restricted precursor; hUBC: human umbilical cord blood cells; NSCs: neural stem cells; hNPC: human neural progenitor cell; hMSC: human mesenchymal stem cell; HSCs: hematopoietic stem cells.
