**5. Stem cell technology for ALS research**

Stem cells are defined as a population of cells that maintains the ability to self-renew and differentiate into several cell types of the adult body. In mammals, several tissues such as muscle, brain and bone marrow, harbor subtypes of stem cells that can give rise to a rela‐ tively small variety of different cell types. These adult stem cells are committed to certain cell lineages and do not produce cells from other tissue types under normal conditions. Un‐ like adult stem cells, embryonic stem cells that can be isolated from the inner cell mass of early stage embryos are pluripotent and can, therefore, still differentiate into virtually any cell type of the human body. While the collection of embryonic stem cells from mice is a widely accepted approach used for disease modeling, the use of human embryonic stem cells is controversial and rises severe ethical concerns. With the discovery that adult human fibroblasts can be reprogrammed to an embryonic stem cell like state, new hope arose for stem cell based approaches in human research. Induced pluripotent stem cells (iPS) are usu‐ ally generated by the introduction of 2-5 defined pluripotency transcription factors into fi‐ broblasts or other readily available differentiated cell types. These transcription factors drastically alter gene expression in the target cells until some of them eventually become pluripotent and can then be isolated and amplified. Initially, the transcription factors were introduced by retroviral or lentiviral constructs leading to the integration of the transgene into the target genome. As the random integration of additional genes can disrupt/alter the expression of endogenous genes, more recent approaches rely on less invasive techniques such as transposons or RNA transfection [74]

genes [65] and downregulation of Bcl-2 [67] and might, therefore, be used to investigate the

Recently, Nardo et al. performed proteomic analysis of PBMCs isolated from 60 sporadic ALS patients and 30 healthy controls [68]. The authors identified and validated in a second cohort 14 protein biomarkers, that could discriminate between ALS patients and controls re‐ gardless of age and gender. Remarkably, of these 14 biomarkers, 5 were able to discriminate between ALS patients and individuals with other neuropathies and 3, among which TDP43, were markers of disease severity. Notably, these results are consistent with a CSF biomarker study reporting that TDP43 levels were increased in ALS patients [69]. The value of this re‐ sult goes beyond the finding of a disease biomarker, as it supports the even more interesting hypothesis that TDP43 could be a common player in early disease in familial as well as sporadic ALS cases. This would confirm what is already suggested by the presence of

Although there is still no consensus on valid biomarkers for ALS [70], proteome analysis has recently led to the identification of fetuin-A and transthyretin (TTR) as candidates to distin‐ guish ALS patients with rapid versus slow disease progression. The upregulation of TTR and fetuin-A, involved in immune regulatory functions, could be associated with the inflam‐ matory state of the CNS. At present, these markers were tested in two independent cohorts of 18 and 20 patients with a follow up of 2 years [71] and TTR had already been identified as a potential biomarker for ALS compared to controls in a previous study [72]. Although fur‐ ther validation is needed, these results are encouraging and would provide an invaluable tool to discriminate between patients with different disease progression rates. This would help clinician determine the timing for clinical intervention such as gastrostomy and non-

Stem cells are defined as a population of cells that maintains the ability to self-renew and differentiate into several cell types of the adult body. In mammals, several tissues such as muscle, brain and bone marrow, harbor subtypes of stem cells that can give rise to a rela‐ tively small variety of different cell types. These adult stem cells are committed to certain cell lineages and do not produce cells from other tissue types under normal conditions. Un‐ like adult stem cells, embryonic stem cells that can be isolated from the inner cell mass of early stage embryos are pluripotent and can, therefore, still differentiate into virtually any cell type of the human body. While the collection of embryonic stem cells from mice is a widely accepted approach used for disease modeling, the use of human embryonic stem cells is controversial and rises severe ethical concerns. With the discovery that adult human fibroblasts can be reprogrammed to an embryonic stem cell like state, new hope arose for stem cell based approaches in human research. Induced pluripotent stem cells (iPS) are usu‐ ally generated by the introduction of 2-5 defined pluripotency transcription factors into fi‐ broblasts or other readily available differentiated cell types. These transcription factors

disease during its progression as well as provide unique biomarkers.

TDP-43 positive aggregates in SALS biopsy samples.

170 Current Advances in Amyotrophic Lateral Sclerosis

**5. Stem cell technology for ALS research**

invasive ventilation [73].

The use of stem cell technologies in ALS research started in 2007 when mouse embryonic stem cells from the most prominent SOD1 model carrying the G93A mutation were estab‐ lished [75]. When differentiated into motor neurons, mild phenotypic differences between motor neurons expressing human wild type SOD1 or the G93A mutation could be observed. After several weeks in culture, SOD1 containing inclusions as well as the overall level of ubiquitinated proteins were more frequent in the motor neurons expressing the mutant hu‐ man protein. In 2009, a human embryonic stem cell line was used to generate motor neurons that were then transfected with different SOD1 mutation containing constructs [76]. The re‐ searchers observed a reduction in neurite length and in line with the study of the mouse G93A embryonic stem cell derived motor neurons, reduced survival. However, in the hu‐ man study, it is unclear whether the observed phenotype arises from the mutations or the increase of SOD1 abundance itself, as a control overexpressing wild type SOD1, was not generated.

Since the discovery of the iPS technology, huge efforts were put in the generation of patient specific iPS lines. Up to now, several hundred lines with various mutations have been gener‐ ated and some are now becoming commercially available thereby getting accessible to a broad scientific community.

In 2008, Dimos et al reported the successful generation of motor neurons and glial cells from an ALS patient derived iPS line carrying a SOD1 mutation causing a mild disease phenotype [14]. Surprisingly, unlike the previous studies with mouse and human embryonic stem cells, no disease related phenotype was reported from these cells until now. This potential lack of phenotype could in part be explained by the patient's late onset and mild disease form. An‐ other report from 2011, where motor neurons were generated from a patient harboring a VAPB mutation, did also not mention any phenotype despite reduced levels of VAPB. As the levels of this protein were already reduced in the fibroblasts used for the reprogram‐ ming, the lower levels in the resulting motor neurons could either be due to mutation in‐ duced expression or translational changes or could also be explained by an incomplete reprogramming of this genomic locus in the generated iPS lines [77]. Recently, the first re‐ port of an iPS line harboring a TDP-43 mutation was published [78]. The motor neurons generated from this line showed elevated TDP-43 levels, but no change in localization or signs of aggregate formation. In addition, motor neurons from both, control and TDP-43 mutant were phenotypically and functionally similar despite an elevated sensitivity to PI3K signaling inhibition and elevated cell death. These data suggest that the toxicity of this TDP-43 mutation might arise from its increased stability leading to a higher overall protein amount in the cell.

Despite the growing number of iPS lines from ALS patients available, no further reports of disease relevant phenotypes or major discoveries of disease mechanisms from the use of these cells were reported so far. In addition, observations from other neurological disorders show a similar trend: cells differentiated from embryonic stem cells or induced pluripotent stem cells often reflect only certain aspects of the disease and sometimes it takes several weeks before such differences can be recorded and/or the observed symptoms are very mild [79, 80]. The problems to reproduce patient phenotypes have ameliorated the initial excite‐ ment about this new method to model neurological diseases. More recently, it became clear that even cells differentiated from individual embryonic stem cell clones or iPS clones from the same patient can show substantial phenotypic differences, a phenomenon called clonal variation [81]. Similar observations were made in a study using iPS lines of ALS patients carrying TDP-43 mutations. After differentiation of individual iPS clones from the same pa‐ tient into motor neurons, the levels of TDP-43 expression as well as aggregate formation and oxidative stress induced cell death showed substantial variation [82]. Considering the con‐ fusing reports, it has to be assumed that the reprogramming as well as the following differ‐ entiation mechanisms are not yet fully under control and more mechanistic research and standardization of the protocols will hopefully soon lead to more pronounced and reprodu‐ cible results. Initial steps to improve reproducibility and differentiation are already under way. The main focus lies on the standardization of the initial characterization of the iPS clones prior to use as well as the improvement of differentiation protocols by addition of various small molecules and growth factors [83, 84].

the other hand, the stem cells can be used for cell replacement approaches. For patients with known mutations, the cells can be genetically modified or corrected prior to reprogramming and differentiation. Such individualized strategies would allow the use of the patient's own

The Use of Human Samples to Study Familial and Sporadic Amyotrophic Lateral Sclerosis: New Frontiers…

http://dx.doi.org/10.5772/56487

173

In ALS, a major future goal would be to produce and replace dying motor neurons. A proof of principle that motor neuron transplantation might become possible came from the obser‐ vation that mouse embryonic stem cell derived motor neurons transplanted into the lumbar part of paralysed adult rats can actually survive and form functional neuromuscular junc‐ tions leading to phenotypical improvements [85]. However, in human ALS patients, the re‐ placement of motor neurons might be more complicated due to the size differences, amount of cells needed and distance that the axon would have to grow out. In addition, it becomes more and more evident that ALS is a non-cell autonomous disease in which astrocytes, mi‐ croglia and oligodendrocytes play a crucial role in modulating disease onset and progres‐ sion [3, 15, 86]. In this context, a strategy approaching several cell types at a time might be more successful than bringing in healthy motor neurons alone into a heavily diseased envi‐ ronment. A promising candidate that can generate various cell types in the CNS and at the same time positively stimulate the neuronal environment by producing neuroprotective fac‐ tors, are neuronal progenitor cells (also called neuronal stem cells). Several reports indicate that transplanted neuronal progenitor cells are able to differentiate into different cell types *in vivo* [87, 88]. Further, injection of NPCs has been shown to ameliorate disease progression in ALS rodent models even if most of the cells do not migrate or differentiate into other cell types in various neurodegenerative diseases [89]. However, it is not known how these cells

cells for transplantation, thereby reducing the risk of graft rejection.

would behave and survive when transplanted into a diseased environment.

to generate enough cells for therapeutic applications.

be safer.

One of the largest limitations for cell replacement strategies to date is the lack of efficiency as well as specificity during the amplification and differentiation step. The differentiation of ES or iPS cells into various different neurons or neuronal progenitor cells is guided by the application of different growth factors and small molecules. However, this process usually generates a mixed population containing many different cell types. The cell type of interest often represents only 30% or even less of the total population. Therefore, it might be difficult

When using ES or iPS lines to generate the cell type of interest, a further drawback is that a small portion of cells remains undifferentiated and immature, thereby representing a major risk factor for transplantation [90]. When neuronal progenitor cells derived from mouse iPS cells were injected into adult mouse brains, they formed tumors in up to 60% of the injected animals [88]. The use or more restricted cell types such as NPCs on the opposite, appears to

Finally, the generation and maintenance of a stable iPS lines from human adult cells is ex‐ pensive and very time consuming [74]. If clinical applications are considered, a thorough characterization of several individual clones needs to be undertaken prior to use, making a widespread application of this approach today unlikely. Very recent reports indicate that fi‐ broblasts can be directly differentiated into several types of neurons and even neuronal pro‐ genitor cells in a much faster and more efficient way than through iPSing [91]. It remains to

While these approaches emerge, sporadic and familial forms of ALS can be modeled with cells isolated from human post mortem spinal cord or brain samples. A recent report dem‐ onstrated that post-mortem isolated neuronal progenitor cells from patients with sporadic or familial ALS, can be differentiated into astrocyte-like cells *in vitro*. Astrocytes from pa‐ tients, but not from healthy controls conveyed toxicity to wild type mouse motor neurons in a co-culture [15]. This system provides a promising tool for testing of potential therapeutic approaches.

#### **5.1. The promises and limitations of stem cells for therapeutic approaches**

The disease modeling described in the upper section is in particular important for the huge proportion of ALS cases, where no causing mutation is known. Until the discovery of the iPS technology, this lack of knowledge made it impossible to model such cases *in vitro* in a cell based assay or with animal models, unless post-mortem cells could be collected. Now we can use skin fibroblasts or other readily available cell types from affected patients during different time points of disease progression prior to end stage. From these fibroblasts, vari‐ ous cell types that are known to play a crucial role in ALS can be generated and their behav‐ ior and interplay in a cell culture dish can be studied in-depth.

Despite emerging into an invaluable tool to study disease mechanisms, stem cells also hold a huge potential for the development of therapeutic approaches for ALS and many other neurodegenerative diseases. On the one hand, the generated cells can now be used in drug screenings to identify new target mechanisms or to assess potential new therapeutics. On the other hand, the stem cells can be used for cell replacement approaches. For patients with known mutations, the cells can be genetically modified or corrected prior to reprogramming and differentiation. Such individualized strategies would allow the use of the patient's own cells for transplantation, thereby reducing the risk of graft rejection.

Despite the growing number of iPS lines from ALS patients available, no further reports of disease relevant phenotypes or major discoveries of disease mechanisms from the use of these cells were reported so far. In addition, observations from other neurological disorders show a similar trend: cells differentiated from embryonic stem cells or induced pluripotent stem cells often reflect only certain aspects of the disease and sometimes it takes several weeks before such differences can be recorded and/or the observed symptoms are very mild [79, 80]. The problems to reproduce patient phenotypes have ameliorated the initial excite‐ ment about this new method to model neurological diseases. More recently, it became clear that even cells differentiated from individual embryonic stem cell clones or iPS clones from the same patient can show substantial phenotypic differences, a phenomenon called clonal variation [81]. Similar observations were made in a study using iPS lines of ALS patients carrying TDP-43 mutations. After differentiation of individual iPS clones from the same pa‐ tient into motor neurons, the levels of TDP-43 expression as well as aggregate formation and oxidative stress induced cell death showed substantial variation [82]. Considering the con‐ fusing reports, it has to be assumed that the reprogramming as well as the following differ‐ entiation mechanisms are not yet fully under control and more mechanistic research and standardization of the protocols will hopefully soon lead to more pronounced and reprodu‐ cible results. Initial steps to improve reproducibility and differentiation are already under way. The main focus lies on the standardization of the initial characterization of the iPS clones prior to use as well as the improvement of differentiation protocols by addition of

While these approaches emerge, sporadic and familial forms of ALS can be modeled with cells isolated from human post mortem spinal cord or brain samples. A recent report dem‐ onstrated that post-mortem isolated neuronal progenitor cells from patients with sporadic or familial ALS, can be differentiated into astrocyte-like cells *in vitro*. Astrocytes from pa‐ tients, but not from healthy controls conveyed toxicity to wild type mouse motor neurons in a co-culture [15]. This system provides a promising tool for testing of potential therapeutic

The disease modeling described in the upper section is in particular important for the huge proportion of ALS cases, where no causing mutation is known. Until the discovery of the iPS technology, this lack of knowledge made it impossible to model such cases *in vitro* in a cell based assay or with animal models, unless post-mortem cells could be collected. Now we can use skin fibroblasts or other readily available cell types from affected patients during different time points of disease progression prior to end stage. From these fibroblasts, vari‐ ous cell types that are known to play a crucial role in ALS can be generated and their behav‐

Despite emerging into an invaluable tool to study disease mechanisms, stem cells also hold a huge potential for the development of therapeutic approaches for ALS and many other neurodegenerative diseases. On the one hand, the generated cells can now be used in drug screenings to identify new target mechanisms or to assess potential new therapeutics. On

**5.1. The promises and limitations of stem cells for therapeutic approaches**

ior and interplay in a cell culture dish can be studied in-depth.

various small molecules and growth factors [83, 84].

172 Current Advances in Amyotrophic Lateral Sclerosis

approaches.

In ALS, a major future goal would be to produce and replace dying motor neurons. A proof of principle that motor neuron transplantation might become possible came from the obser‐ vation that mouse embryonic stem cell derived motor neurons transplanted into the lumbar part of paralysed adult rats can actually survive and form functional neuromuscular junc‐ tions leading to phenotypical improvements [85]. However, in human ALS patients, the re‐ placement of motor neurons might be more complicated due to the size differences, amount of cells needed and distance that the axon would have to grow out. In addition, it becomes more and more evident that ALS is a non-cell autonomous disease in which astrocytes, mi‐ croglia and oligodendrocytes play a crucial role in modulating disease onset and progres‐ sion [3, 15, 86]. In this context, a strategy approaching several cell types at a time might be more successful than bringing in healthy motor neurons alone into a heavily diseased envi‐ ronment. A promising candidate that can generate various cell types in the CNS and at the same time positively stimulate the neuronal environment by producing neuroprotective fac‐ tors, are neuronal progenitor cells (also called neuronal stem cells). Several reports indicate that transplanted neuronal progenitor cells are able to differentiate into different cell types *in vivo* [87, 88]. Further, injection of NPCs has been shown to ameliorate disease progression in ALS rodent models even if most of the cells do not migrate or differentiate into other cell types in various neurodegenerative diseases [89]. However, it is not known how these cells would behave and survive when transplanted into a diseased environment.

One of the largest limitations for cell replacement strategies to date is the lack of efficiency as well as specificity during the amplification and differentiation step. The differentiation of ES or iPS cells into various different neurons or neuronal progenitor cells is guided by the application of different growth factors and small molecules. However, this process usually generates a mixed population containing many different cell types. The cell type of interest often represents only 30% or even less of the total population. Therefore, it might be difficult to generate enough cells for therapeutic applications.

When using ES or iPS lines to generate the cell type of interest, a further drawback is that a small portion of cells remains undifferentiated and immature, thereby representing a major risk factor for transplantation [90]. When neuronal progenitor cells derived from mouse iPS cells were injected into adult mouse brains, they formed tumors in up to 60% of the injected animals [88]. The use or more restricted cell types such as NPCs on the opposite, appears to be safer.

Finally, the generation and maintenance of a stable iPS lines from human adult cells is ex‐ pensive and very time consuming [74]. If clinical applications are considered, a thorough characterization of several individual clones needs to be undertaken prior to use, making a widespread application of this approach today unlikely. Very recent reports indicate that fi‐ broblasts can be directly differentiated into several types of neurons and even neuronal pro‐ genitor cells in a much faster and more efficient way than through iPSing [91]. It remains to be evaluated to which extent these cells recapitulate neurodegenerative disease phenotypes, although a first report from fibroblast derived neurons from a familial Alzheimer's patient seem promising [92].

that anacardic acid had protective effects against arsenite-induced motor neuron death and was able to decrease TDP-43 cytoplasmic aggregates as well as increase neurite length [82]. A different approach was taken in 2011 by Haidet-Phillips and colleagues [15], producing cells from patients without the use of viral vectors or induction of major epigenetic modi‐ fications. In this study, astrocytes were derived from NPCs isolated from ALS patients and it was observed that, regardless of their familial or sporadic origin, these cells were toxic to wild type murine motor neurons expressing GFP under HB9 promoter [15]. The authors found that SOD1 knockout via shRNA could rescue motor neurons at different extents depending on whether these were co-cultured on astrocytes from familial or sporadic cases. This study overcomes some of the major issues related to iPS cells and sets the premises for drug and shRNA screening to target pathways and single genes in‐

The Use of Human Samples to Study Familial and Sporadic Amyotrophic Lateral Sclerosis: New Frontiers…

http://dx.doi.org/10.5772/56487

175

Concluding, it is clear that in the past five years the ALS field has seen a major change of scenario, where more tools are available to study more forms of FALS as well as the striking majority of SALS. As the recent genetic discoveries have highlighted the importance of pre‐ viously unexplored pathways, i.e. RNA metabolism, also common targets linking sporadic and familial ALS have been identified, i.e. TDP-43 and SOD-1. Moreover, the advances in highthroughput screening technology with the advent of new gene profiling techniques, i.e. deep-sequencing, and high content imaging systems are bound to determine the beginning

, Kathrin Meyer and Brian Kaspar

Research Institute at Nationwide Children's Hospital, Columbus, OH, USA

eral sclerosis. Nature reviews. Neurology, 2011. 7(11): p. 616-30.

degeneration. Nature, 2012. 487(7408): p. 443-8.

[1] Ferraiuolo, L., et al., Molecular pathways of motor neuron injury in amyotrophic lat‐

[2] Ilieva, H., M. Polymenidou, and D.W. Cleveland, Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. The Journal of cell biology, 2009.

[3] Lee, Y., et al., Oligodendroglia metabolically support axons and contribute to neuro‐

[4] Geser, F., V.M. Lee, and J.Q. Trojanowski, Amyotrophic lateral sclerosis and fronto‐ temporal lobar degeneration: a spectrum of TDP-43 proteinopathies. Neuropatholo‐ gy : official journal of the Japanese Society of Neuropathology, 2010. 30(2): p. 103-12.

volved in astrocyte toxicity.

of a new era for ALS research.

187(6): p. 761-72.

**Author details**

Laura Ferraiuolo\*

**References**

In summary, the technique of reprogramming holds great promises in terms of disease mod‐ eling and unraveling of underlying mechanisms of sporadic neurodegenerative diseases such as ALS. Despite the current confusion due to the various methods used to generate the lines, the observed clonal variations as well as the limited reflection of disease phenotypes, the field has advanced with tremendous speed if one considers that the first report about reprogramming of mouse fibroblasts was published only 6 years ago. With combined efforts and improved methods, a better understanding and control of the reprogramming mecha‐ nisms can be achieved, thereby facilitating the interpretation and usage of the generated cells.
