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

444 Amyotrophic Lateral Sclerosis

reduce microgliosis; increased lifespan; delayed disease progrssion

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; no

innervations of motor neuron to the skeletal muscle end plates, no effect on ipsilateral hind limb function.

Transplanted cells survive within host skeletal muscles and release GDNF; significant increase in neuromuscular junctions; improves motor neuron survival

continued

patients i.v. injection No clinical benefits Appel et al. 2008

Decelerated linear decline of the forced vital capacity and of the ALS-FRS score in some patients

lives 47 months more than the control group Garbuzova-Davis et al. 2008

Suzuki et al.

Suzuki et al.

Mazzini et al.

Martinez et al.

2010

2009

2008

2007

**Cell type Subject Injection Site Effect Paper** 

Unilateral lumbar spinal cord injection

G93A mice i.v. injection

G93A rats Skeletal muscles

multiple thoracic spinal cord injection

bilateral injection into frontal motor

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:

cortex

human mesenchymal stem cell; HSCs: hematopoietic stem cells.

hUBC hSOD1-

hNPC-GDNF hSOD1-

hMSC-GDNF hSOD1-

ALS

ALS patients

ALS patients

CD34+ HSCs, HLA-matched sibling donors

Autologous bone marrow derived MSCs

Autologous CD133+ cells G93A rats

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.

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

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 improvements ever observed in familial ALS model rats.

Stem Cell Application for Amyotrophic Lateral Sclerosis: Growth Factor Delivery and Cell Therapy 447

prolonging survival, as the two greatly complement each other. Finally, we are now convinced that injections of stem cells in multiple sites are needed in order to alleviate symptoms of ALS. There should be at least one injection that focuses on protecting cell bodies of motor neurons and another that aims to maintain neuromuscular connections. To sum up, stem cell applications have made a lot of contributions to ALS research and have

Fig. 1. Schematic illustration of possible stem cell interventions for ALS therapies. These could include: (1) Motor neuron replacement, differentiation of neural progenitor cells to motor neurons and projection to the periphery; (2) Differentiation and replacement of dysfunctional astrocytes; (3) Modulation of immunological environment around the degenerating motor neuron; (4) Trophic/growth factor delivery via stem cells to provide neuroprotective support for the endogenous populations; (5) Local delivery of growth

This work was support by grants from the ALS association, NIH/NINDS (R21NS06104), the

Acsadi G, Anguelov RA, Yang H, Toth G, Thomas R, Jani A, Wang Y, Ianakova E,

Appel SH, Engelhardt JI, Henkel JS, Siklos L, Beers DR, Yen AA, Simpson EP, Luo Y,

Azzouz M, Ralph GS, Storkebaum E, Walmsley LE, Mitrophanous KA, Kingsman SM,

Mohammad S, Lewis RA & Shy ME. (2002) Increased survival and function of SOD1 mice after glial cell-derived neurotrophic factor gene therapy. *Hum Gene* 

Carrum G, Heslop HE, Brenner MK & Popat U. (2008) Hematopoietic stem cell transplantation in patients with sporadic amyotrophic lateral sclerosis. *Neurology*.

Carmeliet P & Mazarakis ND. (2004) VEGF delivery with retrogradely transported

great potential to bring breakthroughs to the field in the near future.

factors to support neuromuscular junctions and axon integrity.

*Ther*. 2002 Jun 10; Volume 13(9); Pages 1047-59.

2008 Oct 21; Volume 71(17); Pages 1326-34.

University of Wisconsin Foundation, and the Les Turner ALS Foundation.

**7. Acknowledgment** 

**8. References** 

### **4.3 Future research directions**

From the two sets of experiments described in this section, we can conclude that stem cell delivery of growth factors is an effective strategy for ALS treatment. We also know that different sets of delivery tools are needed for the motor neuron cell bodies in the spinal cord and their synaptic connections to the skeletal muscles. Our current knowledge leads us to an initial thought for future development of the field of ALS growth factor/stem cell therapy. Motor neuron cell body protection will be provided by stem cell derived wild type astrocytes and microglia (from hNPC for example); while synaptic/axonal protection will be provided by stem cell derived myoblasts (from hMSC for example). Those cells will be genetically modified to enhance delivery of neurotrophic factors. Lastly, GDNF is only one of the many neurotrophic factors that showed to have beneficial effect on ALS rodent models as mentioned in Section 2 of this chapter. We expect there will soon be tests on the other neurotrophic factors.
