Stem cell therapy for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) (or Lou-Gehrig’s disease) represents a neurodegenerative
disorder characterized by progressive degeneration of motor neurons and its symptoms
including muscle atrophy, weakness, fasciculation, and spasticity 1]. The condition is the most common motor neuron disease, with a worldwide incidence
of 2–3 per 100,000 and a prevalence at 4–6 per 100,000 2], posing a heavy burden on both the families involved and society at large. Patients
tend to die 3–5 years after diagnosis due to progressive motor neuron loss and weakness
of skeletal muscles, especially those muscles responsible for breathing, which is
the primary cause of death caused by ALS 3]. The pathogenesis of ALS is believed to be multifactorial. For the familial forms,
several genetic mutations have been identified as being associated with the disease,
including mutations in Cu superoxide dismutase (SOD1), TAR DNA binding protein-43
(TDP-43), the C9orf72 gene (the most common mutation underlying familial forms of
ALS), and the recently discovered TBK1 gene encoding a protein involved in two essential
cellular pathways of emerging interest in ALS research: autophagy and inflammation
4]. In the more common forms of sporadic ALS, neurodegeneration might result from an
intricate interaction among multiple cell types and several different mechanisms,
including protein aggregation, glutamate-mediated excitotoxicity, mitochondrial dysfunction,
oxidative stress, impaired axonal transportation, altered glial cell function, and
deficiency of neurotrophic factors 5]. All of these factors can eventually lead to the disruption of axonal transport processes
via intracellular accumulation of neurofilaments 3], 6]. This heterogeneity of ALS makes it difficult to identify the exact cause of ALS
and so develop effective therapies. Except Riluzole, which is believed to be able
to extend survival by a few months 7], to date, few treatments have proved to be highly or consistently effective 8].

Stem cell therapy is a promising potential treatment option for ALS, given stem cells’
remarkable plasticity and ability to differentiate into multiple neuronal lineages
9]; they are consequently a valuable source for replacement cell therapy. When locally
or systemically transplanted, stem cells are capable of migrating to disease-associated
loci to exert the desired therapeutic effect 10]. Currently available cell therapies may take advantage of a variety of stem cells
to modify disease pathophysiology 11], slow down or even halt the progression of disease, possibly by providing protective
factors to surrounding cells, modulating the host immune environment, inhibiting inflammation,
or even replacing injured cells 12]–17]. Several types of stem cells have been studied as possibilities for treating ALS,
including neural stem cells (NSCs), mesenchymal stem cells (MSCs), glial-restricted
progenitor cells (GRPs), embryonic stem cells (ESCs), and induced pluripotent stem
cells (IPSCs) 18]. Here, we will comprehensively review the current state of research concerning treatments
for ALS using stem cells and provide information on aspects of further research into
stem cell-based therapies for ALS.

Neural stem cells

NSCs originate from the neuroectoderm of early embryos and are found in embryonic,
fetal, and adult nervous systems. They possess the potential to differentiate into
any cell type in the central nervous system (CNS) (although NSCs derived from adult
tissues show a more limited differentiation capacity 19]). The integration ability and prospective therapeutic efficacy of human neural stem
cells (hNSC) has been demonstrated in rodent models of neurological diseases 20]–23]. Apart from regenerating lost neuronal cells, NSCs can also improve the functional
outcomes of rats through auxiliary mechanisms, such as neurotrophism 24]–26] and immunosuppression 27]–29].

A number of studies have demonstrated that NSC therapy had beneficial effects on ALS
rats 17], 30]. Transplanted NSCs could differentiate into neurons and form synaptic connections
with host tissues, delay disease onset and progression, and prolong the survival of
experimental animals 17]. Hefferan et al. found that grafted hNSCs protected adjacent motor neurons and helped
to achieve transient functional improvement 31], and they speculated that this transient functional improvement was attained possibly
because transplanted NSCs elicited neurogenesis and triggered intrinsic repair mechanisms
in the spinal cord 32]. More encouragingly, Teng and co-workers found that besides a delay in disease progression
and an improvement in motor function, a quarter of the NSC-grafted ALS mice survived
three times longer than their non-grafted counterparts 33].

Given the pre-clinical support for NSC-based therapies, in 2009, the FDA approved
a clinical trial on the safety and tolerability of surgical delivery of stem cells
and any resulting cell toxicity 34]. A total of 18 patients with ALS received an intraspinal fetal-derived NSC (NSI-566RSC)
engraftment following a risk escalation paradigm, progressing from non-ambulatory
to ambulatory subjects, lumbar to cervical spinal cord segments, and unilateral to
bilateral injections across five cohorts. After monitoring the patients for 2.5 years,
all patients tolerated the procedure without major surgical complications, such as
injection-attributable neurological worsening, and there were no indications that
the stem cells themselves were either toxic or injurious to the spinal cord. In an
expansion of the above study using NSCs isolated from human fetal spinal cord tissues,
Mazzini et al. transplanted human fetal brain tissues into the anterior horns of the
spinal cord and additionally used a much higher cell dosage and a milder immunosuppression
regimen 35]. They verified the safety and tolerability by clinical assessment against safety
measures and follow-up, utilizing neuroimaging and other techniques 35]. These studies have paved the way for future clinical trials on the efficacy and
dosage of NSC treatment for ALS. A phase I clinical trial that began in July 2011
is designed to verify the safety of expanded hNSCs and microsurgery and to evaluate
their effect on the quality of life of the patients (ClinicalTrials.gov Identifier:
NCT01640067). A phase II clinical trial, which started in May 2013, is aiming to assess
the feasibility, safety, toxicity, and maximum tolerated (safe) dose of the NSC treatment
(ClinicalTrials.gov Identifier: NCT01730716).

However, in addition to two issues which hamper NSC studies, namely ethical issues
and immune rejection problems, NSCs are derived from fetal spinal cord (NSI-566RSC)
36] or fetal brain tissues 35], two sources of cells that are very limited. Consequently, their large-scale use
in clinical trials remains a challenge.

Mesenchymal stromal cells

MSCs are multipotent adult stem cells that can be easily extracted from various adult
connective tissues (i.e., bone marrow and adipose tissue) and can differentiate into
a variety of cells 37]–39].

A number of studies employing animal models of ALS have investigated the therapeutic
potential of MSCs by injecting cells either peripherally or directly into the spinal
cord. Marconi et al. assessed the efficacy of the systemic administration of adipose-derived
mesenchymal stem cells (ASC) in SOD1-mutant mice and found that the cells not only
significantly delayed motor deterioration for 4–6 weeks and maintained the number
of motor neurons but also up-regulated the levels of glial-derived neurotrophic factor
(GDNF) and basic fibroblast growth factor (bFGF) in the spinal cord. Given that ASCs
produce bFGF but not GDNF, these findings indicated that ASCs may promote neuroprotection
either directly and/or by modulating the response of local glial cells toward a neuroprotective
phenotype 40]. Similarly, intramuscular transplantation of MSCs engineered to secrete GDNF was
found to attenuate motor neuron loss and prolong the lifespan of ALS rats 41]. In another study, MSCs were genetically modified to release GDNF or VEGF, and when
injected into animals, they extended survival and alleviated the loss of motor function
42]. The therapeutic effect of MSCs may primarily capitalize on innate trophic support
from themselves or from the delivery of augmented growth factors. Intraspinal, intracerebral,
intrathecal, and intravenous injection of autologous MSCs in SOD1-G93A mice have also
reported beneficial effects on disease progression, including slowed loss of motor
neurons, improved motor function, and extended survival 43]–48]. Given the fact that MSCs can deliver neurotrophic, anti-inflammatory, and immunomodulatory
molecules 49], 50], these cells are a promising treatment approach for ALS.

In 2003, Mazzini and colleagues conducted the first clinical studies to determine
the safety and tolerability of direct intraparenchymal transplantation of MSCs for
the treatment of ALS. MSCs were isolated from allogeneic ALS patients’ bone marrow
aspirates and transplanted into the thoracic spinal cord. While there was no functional
improvement following MSC transplantation, no serious adverse effects and no detrimental
effects on neurological function were reported 51]. Their follow-up studies, lasting more than 4 years after surgery, revealed no signs
of toxicity or abnormal cell growth and showed that four patients might have benefited
from the treatment 52], 53]. Subsequently, a number of clinical trials have evaluated autologous MSC transplantation
and demonstrated that intraspinal, intrathecal, and intracerebral transplants were
safe and feasible 54]. It is worth mentioning that three clinical studies innovatively mobilized endogenous
MSCs by using granulocyte-colony stimulating factor (G-CSF) in ALS patients, and their
MSCs were instantaneously increased and no major adverse events were found in any
of the three studies 55]–57]. Moreover, BrainStorm Cell Therapeutics developed a cell type trademarked as “NurOwn™”
for the treatment of ALS. The cells can differentiate into specialized neuron-supporting
cells capable of stably secreting neurotrophic factors (MSC-NTFs). Currently, a phase
II clinical trial using NurOwn cells began in 2014 to evaluate the safety and efficacy
of the cells (ClinicalTrials.gov Identifier: NCT02017912). Another phase II study
using NurOwn cells, this time in a dose-escalating clinical study, is now under way
(ClinicalTrials.gov Identifier: NCT01777646).

MSCs can be relatively easily obtained from adult tissues, and their application does
not pose substantial ethical issues 58], 59], and because ALS does not influence MSC expansion and differentiation 60], the cells can be extracted from patients themselves, thus avoiding immune rejection.
So, MSCs seem to be an attractive candidate for ALS cell therapy. However, deriving
MSCs from either bone marrow or adipose tissue causes, to some degree, trauma. What
is more, MSCs are of mesodermal origin and thus their ability to transdifferentiate
into neuronal cells of ectodermal origin is questionable 61]. And as far as we know, so far, there are no robust pre-clinical studies on the long-term
safety, in vivo differentiation, dosage, and biological activity of human MSCs used
for the treatment of ALS. Therefore, studies on its further application in clinical
practice are warranted.

Glial-restricted progenitor transplantation

Many studies have looked into the role of astrocytes in ALS and demonstrated that
astrocytes derived from SOD1 mice and ALS patients could induce motor neuron death,
possibly through a Bax-dependent mechanism triggered by toxic soluble factors (termed
“gliotransmitters”) 62]–66]. When astrocytes derived from SOD1 glial progenitors were transplanted into mice,
they could induce host motor neuron death, focal weakness of the corresponding limb,
and gliosis of host astrocytes and microglia 67], 68]. Recent research has demonstrated that human glial progenitor transplantation and
gene expression was independent of the ALS neurodegenerative spinal cord environment
69], indicating that some cell autonomous changes take place in astrocytes expressing
ALS-linked mutations and treatment of ALS with astrocytes is feasible.

GRPs are the earliest progenitor cell type derived from the embryonic spinal cord,
and they show a tripotential phenotype in their ability to differentiate to oligodendrocytes
and two types of astrocytes 70], 71]. Lepore et al. isolated GRPs from the rat embryonic spinal cord and transplanted
them into the cervical spinal cord of SOD1-G93A rats. After injection, they found
that the grafted cells survived in the diseased tissues and differentiated efficiently
into astrocytes, with microgliosis alleviated at the transplanted sites; additionally,
survival was extended, motor neuron loss was ameliorated and declines in forelimb
motor capability slowed, and respiratory functions improved 72]. Later, another study conducted by the same research team showed that hGRP (also
referred to as Q-Therapeutics’ Q-Cells®) derived from human fetal forebrain robustly
survived and migrated into both gray and white matter and differentiated into astrocytes
in SOD1-G93A mice spinal cord. However, Q-Cells engraftment did not lead to motor
neuron protection or any therapeutic benefits in terms of functional outcome measures
73]. The discrepancies between the two GRP transplantation studies may be due to the
differences in cell types (allograft versus xenograft, rat spinal versus human forebrain-derived),
cell maturity, the number of injection sites, and transplanted cells 74]. As the functions of astrocytes vary depending upon their origins 75], the conflicting results suggest to us that further research should be conducted
to understand the influence of cell type and cell number on clinical outcome.

Like hNSCs, hGRPs are derived from fetal forebrain tissues 76], and their widespread clinical application is greatly hampered by the scarcity of
resources, ethical issues, and potential for immune rejection.

Embryonic stem cells

ESCs, derived from the inner cell mass of the blastocyst 77], can be efficiently differentiated into any cell type both in vitro and in vivo,
and these differentiated cells present morphological, biochemical, and physiological
traits similar to their in vivo counterparts. Many important and decisive differentiation
factors have been discovered, and simple protocols for ESC differentiation into motor
neurons are available.

In 2005, Zhang et al., for the first time, reported the successful differentiation
of human ESC (hESC) into a motor neuron (MN) phenotype 78]. Wyatt et al. transplanted hESCs-derived motor neuron progenitors (hMNPs) into three
animal models of motor neuron loss: SMA (?7SMN), ALS (SOD1-G93A), and spinal cord
injury (SCI) 79]. The transplanted cells survived, differentiated, and secreted physiologically active
growth factors in vivo, thereby significantly increasing the number of spared endogenous
neurons. The ability to maintain dying motor neurons by providing motor neuron-specific
neurotrophic support is a powerful potential treatment strategy for ALS.

Though the use of hESCs in treating ALS in animals are encouraging, not all hESC lines
can differentiate into neural lineages, probably because of inherent differences and/or
the underlying genetics of the embryos from which the lines were derived 80], 81]. In addition, hESCs are derived from pre-implantation human embryos 82], and although hESCs can be maintained and expanded indefinitely, their use comes
with significant ethical concerns and potential immune response issues.

Induced pluripotent stem cells

IPSCs can be derived from patients’ somatic cells by reprogramming with specific factors
83]. iPSCs express stem cell markers and have the ability to give rise to all three germ
layers, as these cells are derived from adult somatic tissues they bypass ethical
concerns, and so are promising candidates for stem cell therapy for ALS.

In 2008, Dimos et al. developed the first strain of human iPSCs from an 82-year-old
familial ALS woman 84]. Mitne-Neto and colleagues successfully reprogrammed fibroblasts from an ALS8 patient
into pluripotent stem cells and differentiated them into motor neurons 85]. Popescu et al. showed that iPSC-derived neural progenitors efficiently engrafted
into the adult spinal cord and could survive in high numbers 86]. They postulated that the transplantation of stem cell-derived neural progenitors
might exert a dual beneficial effect: replacing lost motor neurons and serving as
a source of neurotrophic factors and modifiers of the toxic environment. Recently,
a similar study generated and purified a specific NSC population from human iPSCs.
After injection of these cells into SOD1-mutated mice, NSCs engrafted and migrated
into the CNS, resulting in improved neuromuscular function and motor unit pathology
and a significantly prolonged life span 87]. These beneficial effects are believed to be linked to multiple mechanisms, including
production of neurotrophic factors and reduction of microgliosis and macrogliosis,
thus leading to increased resistance to death of motor neurons and neurodegeneration.
In a series of long-term studies, Chen et al. showed iPSC-derived neural progenitors
mostly differentiated into astrocytes, replaced the endogenous astrocytes, formed
networks through their processes, and encircled endogenous neurons 68]. Preclinical studies, currently being conducted at Johns Hopkins University, use
human iPSC-derived glial-restricted precursors (iPSC-GRPs) 88] and may offer perspectives for the use of iPSC-based therapy in ALS 87].

Among all the cells mentioned above, hiPSCs have incomparable advantages over other
cells. Currently, hiPSCs can be obtained from the blood 89] or urine 90] and so are relatively easily available, rendering their use feasible in clinical
treatment. Therefore, hiPSCs are an attractive candidate for ALS cell therapy. A major
concern about the application of hiPSCs might be its potentially high tumor-forming
capability.  However, in actual research of hiPSCs, tumor formation was rare 68], 86], 91], 92], except for a very small proportion of grafts that remained positive for neural progenitor
marker genes 91]. If the number and the differentiation of transplanted neural progenitor cells are
well controlled, the possibility for tumor formation can be substantially decreased.
Additionally, concerns about random viral integration could be ameliorated by the
development and optimization of the use of episomal plasmids, recombinant proteins
with membrane permeable peptides, or Sendai virus vectors.