Neural stem cell-based treatment for neurodegenerative diseases

Authors

  • Seung U. Kim,

    Corresponding author
    1. Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea
    • Division of Neurology, Department of Medicine, UBC Hospital, University of British Columbia, Vancouver, British Columbia, Canada
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  • Hong J. Lee,

    1. Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea
    2. Division of Neurology, Department of Medicine, UBC Hospital, University of British Columbia, Vancouver, British Columbia, Canada
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  • Yun B. Kim

    1. Department of Toxiology, Chungbuk National University School of Vetrinary Medicine, Cheungju, Korea
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Correspondence: Seung U. Kim, MD, PhD, Division of Neurology, Department of Medicine, UBC Hospital, University of British Columbia, Vancouver, BC, Canada V6T 2B5. Email: sukim@mail.ubc.ca

Abstract

Human neurodegenrative diseases such as Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) are caused by a loss of neurons and glia in the brain or spinal cord. Neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and stem cell-based cell therapies for neurodegenerative diseases have been developed. A recent advance in generatioin of a new class of pluripotent stem cells, induced pluripotent stem cells (iPSCs), derived from patients' own skin fibroblasts, opens doors for a totally new field of personalized medicine. Transplantation of NSCs, neurons or glia generated from stem cells in animal models of neurodegenrative diseases, including PD, HD, ALS and AD, demonstrates clinical improvement and also life extension of these animals. Additional therapeutic benefits in these animals can be provided by stem cell-mediated gene transfer of therapeutic genes such as neurotrophic factors and enzymes. Although further research is still needed, cell and gene therapy based on stem cells, particularly using neurons and glia derived from iPSCs, ESCs or NSCs, will become a routine treatment for patients suffering from neurodegenerative diseases and also stroke and spinal cord injury.

Introduction

Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for human neurological diseases. However, the paucity of suitable cell types for cell therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out.

Stem cells are defined as cells that have the ability to renew themselves continuously and possess pluripotent ability to differentiate into many cell types. Two types of mammalian pluripotent stem cells, ESCs derived from the inner cell mass of blastocysts and embryonic germ cells (EGCs) obtained from post-implantation embryos, have been identified and these stem cells give rise to various organs and tissues.[1, 2] Recently there has been an exciting development in generation of a new class of pluripotent stem cells, iPSCs, from adult somatic cells such as skin fibroblasts by introduction of embryogenesis-related genes.[3, 4] A recent study has indicated that patients' own fibroblasts could directly be converted into neurons by combinatorial expression of three neural lineage-specific transcription factors, Ascl1, Brn2 and Myt1l. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials, and form functional synapses.[5] In another study, a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1 and Ptx3, can directly and effectively reprogram human fibroblasts into dopaminergic (DA) neurons. The reprogrammed cells stained positive for cell type-specific markers for DA neurons.[6]

In addition to ESCs and iPSCs, tissue-specific stem cells could be isolated from various tissues of more advanced developmental stages such as hematopoietic stem cells (HSCs), amniotic fluid stem cells, bone marrow MSCs, adipose tissue-derived stem cells, and NSCs. Among these, existence of multipotent NSCs has been known in developing or adult rodent brain with properties of indefinite growth and potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes.[7-11]

In humans, existence of NSCs with multipotent differentiation capability has also been reported in embryonic and adult human brain.[7, 10, 12] In a group of cancer patients who had infusion of chemical bromodeoxyuridine (BrdU) for diagnostic purposes and later died, evidence that new neurons are continuously being generated in adult human CNS has been demonstrated.[13] In a recent study, multipotent and self-renewing human NSCs were isolated from the adult human spinal cord of organ transplant donors, cultured for many passages and differentiated into neurons and glia following transplantation into spinal cord injured rats.[14] The possible provision of adult human NSCs with unique capacity to expand and potential to differentiate into neurons and glia opens doors for therapeutic application of these cells for neurological diseases. However, in practice it is difficult to secure adult human CNS tissues for preparation of adult NSCs, and for this reason stable cell lines of human adult NSCs were developed to serve as a good alternative cellular source.

Continuously dividing immortalized cell lines of NSC have been generated by introduction of oncogenes and these immortalized NSC lines have advantageous characteristics for basic studies on neural development and cell replacement therapy or gene therapy studies: (i) stable immortalized NSC cells are homogeneous since they were generated from a single cell, tha is, single clone; (ii) immortal NSC cells can be expanded readily in large numbers in a short time; and (iii) stable expression of therapeutic genes can be achieved readily.[6, 10, 15-17] Immortalized NSCs have emerged as a highly effective source for genetic manipulation and gene transfer into the CNS ex vivo; immortalized NSCs were genetically manipulated in vitro, survive, integrate into host tissues and differentiate into both neurons and glial cells after transplantation to the intact or damaged brain in vivo.

We have previously generated immortalized cell lines of human NSCs by infecting fetal human brain cells grown in primary culture with a retroviral vector carrying v-myc oncogene and selecting continuously dividing NSC clones. Both in vivo and in vitro these cells were able to differentiate into neurons and glial cells and populate the developing or degenerating CNS.[6, 10, 11] Cell replacement and gene transfer to the diseased or injured CNS using NSCs have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases, including Parkinson's disease (PD), Huntington's disease (HD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), stroke, spinal cord injury (SCI) and brain tumors.[6, 10, 11, 18, 19] There are still many obstacles to be overcome before clinical application of NSC-based cell therapy in neurologically diseased patients is adopted: (i) it is still uncertain how to generate specific cell types of neurons or glia suitable for cellular grafts in great quantity from NSCs; (ii) it is required to abate safety concerns related to tumor formation following NSC transplantation; and (iii) it needs to be better understood by what mechanism transplantation of NSCs leads to an enhanced functional recovery. Continued and extensive progress in stem cell research in both basic and pre-clinical settings should support the hope for development of NSC-based therapies for neurodegenerative diseases. This review focuses on the utility of stem cells, particularly NSCs, as substrates for structural and functional repair of the diseased or injured brain.

Parkinson's Disease

Parkinson's disease, characterized by an extensive loss of dopamine (DA) neurons in the substantia nigra pars compacta and their terminals in the striatum, affects more than 500 000 people in the US and about 50 000 new cases are reported annually.[20, 21] While the etiology of idiopathic PD is not known, several predisposing factors for the dopamine depletion associated with the disease have been suggested, including programmed cell death, viral infection, and environmental toxins. As an effective treatment for PD, patients have been given L-dihydroxyphenyl alanine (L-DOPA), a precursor of dopamine, but long-term administration of L-DOPA consequently produces grave side effects.[22, 23] More recently, surgical deep brain stimulation has been adopted as a successful treatment for PD patients.[24]

Since the late 1980s, transplantation of human fetal ventral mesencephalic tissues into the striatum of PD patients has been used as a successful therapy for patients with advanced disease.[25-28] However, this fetal tissue transplantation has serious problems associated with ethical and religious questions and logistics of acquiring fetal tissues. In addition, recent reports have indicated that the survival of transplanted fetal mesencephalic cells in the patients' brain was very low and it was difficult to obtain enough fetal tissues needed for transplantation.[29] To circumvent these difficulties, utilization of neurons with dopaminergic (DA) phenotype generated from ESCs, iPSCs, MSCs or NSCs could serve as a practical and effective alternative for the fetal brain tissues for transplantation. DA neurons were generated from mouse ESCs after treatment with fibroblast growth factor 8 (FGF8) and sonic hedgehog,[30, 31] over-expression of Nurr1[32, 33] or Bcl-XL,[34] or co-culture with a mouse bone marrow stromal cell line.[35] Neurons with DA phenotype have been generated from monkey ESCs by co-culturing with mouse bone marrow stromal cells and behavioral improvement was seen in MPTP-lesioned monkeys following intra-striatal transplantation of these cells.[36] DA neurons were also generated from neural progenitor cells derived from fetal brain and induced functional recovery following brain transplantation in parkinsonian monkeys.[37]

Transplantation of NSCs in the brain attenuates anatomic or functional deficits associated with injury or disease in the CNS via cell replacement, the release of specific neurotransmitters, and the production of neurotrophic factors that protect injured neurons and promote neuronal growth. Recently we have generated continuously dividing immortalized cell lines of human NSC from fetal human brain cell culture via a retroviral vector encoding v-myc[10, 17, 38] and one of the immortalized NSC lines, HB1.F3, induced functional improvement in a rat model of PD following transplantation into the striatum.[39]

Earlier studies have used gene transfer technology to develop treatment for PD by transferring the tyrosine hydroxylase (TH) gene, a rate-limiting step enzyme in catecholamine biosynthesis process, into certain cell types and then implant these cells into the brain of PD animal models.[40-42] However, gene transfer of TH using genetically modified cells produced only partial restoration of behavioral and biochemical deficits in PD animal models, since the cells utilized did not carry sufficient amount of tetrahydrobiopterin (BH4), a cofactor to support TH activity.[43] Therefore, it is necessary to transfer additionally guanosine-triphosphate cyclohydrolase-1 (GTPCH-1) gene that is the first and rate-limiting enzyme in the BH4 biosynthetic pathway.[44] Immortalized CNS-derived mouse NSC line C17.2 was transduced to carry the TH gene and GTP cyclohydrorylase-1(GTPCH-1) gene for production of L-DOPA and following intra-striatal implantation behavioral improvement was seen in 6-hydroxydopamine-lesioned rats.[45] We have similarly engineered the HB1.F3 human NSC line to produce L-DOPA by double transduction with cDNAs for human TH and GTPCH-1, and following transplantation of these cells in the brain of a PD rat model led to enhanced L-DOPA production in vivo and induced functional recovery.[46]

Previous studies have reported that mouse or human ESC-derived DA neurons have shown efficacy in PD animal models; however, there are considerable safety concerns for ESCs related to risk of tumor formation and neural overgrowth. More recent studies have indicated that functional human DA neurons could be generated efficiently from human ES cells and upon transplantation in rat PD models ES cell-derived DA neurons induced behavior recovery in the animals.[47-49] In a recent study, investigators generated three lines of mouse DA neurons at three stages of differentiation (early, middle and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Mid-stage neuron (Nurr1 + stage) cell grafts had the greatest amount of DA neuron survival and behavioral improvement in parkinsonian mice.[50] Human DA neurons derived from iPS cells may provide an ideal cellular source for transplantation therapy for PD since they could be generated from patients' own fibroblasts and do not cause immune rejection. However, developing an effective cell therapy approach for PD using iPS cells relies on optimizing in vitro production of iPS cell-derived DA neurons and preventing potential risk of teratoma formation in vivo.

A recent study has reported generation of DA neurons from iPS cells derived from fibroblasts and improved behavior following transplantation of these DA neurons in PD model rats.[51] When multiple human iPSC lines derived by virus- and protein-based reprogramming were compared, DA neurons derived from protein-based iPSCs were best suited for transplantation since they exhibited gene expression, physiological and electrophysiological properties similar to those of human midbrain DA neurons.[52] DA neurons were also generated from iPS cells from PD patients and these DA neurons can be transplanted without signs of neurodegeneration into the PD animal model. The PDiPS cell-derived DA neurons survived at high numbers, and mediated functional effects in PD animals.[53] These PDiPS cell-derived DA neurons could be used for screening new drug development in PD. More recently human fibroblasts were directly converted into DA neuron-like cells by the use of combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1 and Pitx3, and the reprogrammed cells stained positive for various markers for DA neurons.[6] Although further research is still required, cell therapy based on DA neurons derived from iPS cells[54] or DA neurons directly converted from fibroblasts may become a promising treatement for PD patients in the coming years.

A summary of preclinical studies of stem cell transplantation in PD animal models in rat and monkey is shown in Table 1.

Table 1. Stem cell-based cell therapy in experimental Parkinson's disease models
Reference (number)Animal modelTransplanted cellsAdditional treatmentFunctional outcome
  1. 6-OHDA, 6-hydroxydopamine; BMSC, bone marrow mesenchymal stem cell; ESC, embryonic stem cell; GTPCH-1, GTP cyclohydrolyrase-1; iPS cell, Induced pluripotent stem cell; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NPC, neural precursor cell; NSC, neural stem cell; TH, tyrosine hydrpxylase.
Hagell 2002[29]Rat, 6-OHDANPC (rat)FGF8/SHHRotation↓
Kim JH et al. 2002[31]Rat, 6-OHDANSC (rat) – DA neuronNoneNot tested
Takagi et al. 2005[36]Monkey, MPTPESC (monkey)Stromal cell (mouse) feederPFS-Parkinsonian factor score↓
Ryu et al. 2005[45]Rat, 6-OHDAImmortalized NSC (mouse, C17-2)

TH/GTPCH1

Gene transfer

Rotation↓
Kim SU et al. 2006[46]Rat, 6-OHDAImmortalized NSC (human, HB1.F3)

TH/GTPCH1

Gene transfer

Rotation↓
Yasuhara et al. 2006[39]Rat, 6-OHDAImmortalized NSC (human, HB1.F3)NSC migrationRotation↓
Redmond et al. 2007[37]

Monkey

MPTP

NSC (human)NonePFS-Parkinsonian factor score↓
Cho et al. 2008[47]Rat, 6-OHDADA neurons from ES cells (human)None

Rotation↓

Beam walking↑

Wernig et al. 2008[51]Rat, 6-OHDADA neurons from ES cells (human)

Wnt signal

Shh

Rotation↓
Kriks et al. 2011[49]Rat, 6-OHDADA neurons from iPS cells (human)NoneRotation↓

Huntington's Disease

Huntington's disease is an autosomal dominant neurodegenerative disorder characterized by involuntary choreic movements, cognitive impairment and emotional disturbances.[55, 56] Despite identification of the HD gene and associated protein, the mechanisms involved in the pathogenesis of HD remain largely unknown and this hampers effective therapeutic interventions. Transplantation of fetal human brain tissue may serve as a useful strategy in reducing neuronal damage in the HD brain and a recent study has documented improvements in motor and cognition performance in HD patients following fetal cell transplantation.[57] This trial follows previous reports in HD experimental animals that positive effects of fetal striatal cell transplantation to ameliorate neuronal dysfunction[58] and that striatal graft tissue could integrate and survive within the progressively degenerated striatum in a transgenic HD mouse model.[59] The latter study is consistent with results obtained from HD patients indicating survival and differentiation of implanted human fetal tissue in the affected regions.[60] Cell replacement therapy using human fetal striatal grafts has shown clinical success in HD patients. However, a recent study has reported neural overgrowth of grafted tissue in an HD patient who survived 5 years post-transplantaion.[61] Overgrown grafts were composed of neurons and glia embedded in disorganized neurpil. This report recalls safety concerns for fetal cell grafts related to potential risk of neural overgrowth following transplantation in the brain of HD patients.

Transplantation of NSCs to replace degenerated neurons or genetically modified NSCs producing neurotrophic factors have been used to protect striatal neurons against excitotoxic insults.[62] At present, little is known regarding whether implantation of NSCs prior to neuropathological damage could alter the progressive degeneration of striatal neurons and motor deficits that occur in HD. This question is important since the genetic study of HD gene mutation[63] and neuroimaging can provide details on factors involved in the progression of HD,[64, 65] suggesting early intervention using brain transplantation could be effective in “pre-clinical” HD patients carrying the mutant HD gene. We have investigated the effectiveness of proactive transplantation of human NSCs into rat striatum of an HD rat model prior to lesion formation and.demonstrated significantly improved motor performance and increased resistance to striatal neuron damage compared with control sham injections.[66] The neuroprotection provided by the proactive transplantation of human NSCs in the rat model of HD appears to be contributed by brain-derived neurotrophic factor (BDNF) secreted by the transplanted human NSCs.

Rodents and primates with lesions of the striatum induced by excitotoxic kainic acid (KA), or quinolinic acid (QA) have been used to simulate HD in animals and to test efficacy of experimental therapeutics on neural transplantation.[67] Excitotoxic animal models induced by QA, which stimulates glutamate receptors, and resembles the histopathologic characteristics of HD patients, were utilized for cell therapy with mouse embryonic stem cells, mouse neural stem cells, mouse bone marrow mesenchymal stem cells and primary human neural precursor cells, and resulted in varying degrees of clinical improvement.[68-73] We have recently injected human NSCs intravenously in QA-HD model rats and demonstrated functional recovery in HD animals.[72, 73] The systemic transplantation of NSCs via an intravascular route is probably the least invasive method of cell administration.[73] Neural cell transplantation into striatum requires an invasive surgical technique using a stereotaxic frame. Non-invasive transplantation via intravenous routes, if effective in humans, is much more attractive.

Systemic administration of 3-nitropropionic acid (3-NP) in rodents leads to metabolic impairment and gradual neurodegeneration of the basal ganglia with behavioral deficits similar to those associated with HD,[74, 75] and murine and human NSCs have been transplanted in the brain of 3-NP-HD animal models.[66, 76] The compound 3-NP is a toxin which inhibits the mitochondrial enzyme succinate dehydrogenase (SDH) and tricarboxylic acid (TCA) cycle, thereby interfering with the synthesis of ATP.[77]

We have investigated the effectiveness of transplantation of human NSCs into adult rat striatum prior to striatal damage induced by 3-NP toxin.[66] Animals receiving intrastriatal implantation of human NSCs 1 week prior to 3-NP treatments exhibited significantly improved motor performance and increased resistance to striatal neuron damage compared with control sham injections. The neuroprotection provided by the proactive transplantation of human NSCs in the rat model of HD appears to be contributed by brain-derived neurotrophic factor (BDNF) secreted by the transplanted human NSCs. Previous studies have also demonstrated that BDNF could block neuronal injury under pathological conditions in animal models of HD.[78, 79] These findings suggest that proactively transplanted human NSCs were well integrated in the striatum and supported the survival of host striatal neurons against neuronal injury.

To develop an effective stem cell-based cell therapy for HD, it is desirable to use genetic animal models, but earlier studies have used chemical (QA or 3-NP)-induced animal models and only a small number of studies have used transgenic HD animals. In YAC HD transgenic mice, bone marrow MSCs genetically modified to express BDNF were transplanted in striatum and induced behavioral improvement.[80] In another study in R6/2 HD transgenic mice, transplantation of adipose tissue-derived stem cells (ADSCs) improved motor function and increased the survival of striatal neurons.[81]

Human striatal neural stem cell line cells were treated with a hedgehog agonist to generate DARPP-32 cells and transplanted in R6/2 HD transgenic mouse brain. The results were disappointing that the outcome was the same as a vehicle control injection.[82] This study is only one using human NSCs for cell therapy in HD genetic animal model.

Human NSCs derived from ESCs could provide a viable cellular source for cell therapy in HD, since they can be expanded indefinitely and differentiate into any cell type desired. Three previous studies have shown that neurons expressing striatal markers could be induced from ESCs and brain transplantation of these ESC-derived neurons in QA-lesioned rats leads to behavioral recovery in the animals.[83-85]

We have previously written a review that focuses on the stem cell-based therapy for HD and investigators who wish to learn more about the subject are referred to the review article.[86] A summary of preclinical studies of stem cell transplantation in HD animal models is shown in Table 2.

Table 2. Stem cell-based cell therapy in experimental Huntington's disease models
Reference (number)Animal modelTransplanted cellsHistology and lesion volumeFunctional outcome
  1. 3-NP, 3-nitropropionic acid; BBB, blood-brain barrier; BMSC, bone marrow stromal cells; NeuN, neuron-specific nuclear protein; NPC, neural precursor cell; NSC, neural stem cell; NSC, neural stem cell; QA, quinolinic acid.
Kordower et al. 1997[68]Rat, Quinolinic acid/ QANSC (mouse)

Intact BBB

Lesion vol↓

Not tested
Armstrong et al. 2000[69]Rat, QABMSC (mouse)

GAD + cells 0.3%

No change

No improvement
McBride et al. 2004[70]Rat, QANPC (fetal human)

NPC migration

Lesion vol↓

Cylinder test↓
Visnyei et al. 2006[71]Rat, QAESC (mouse)

NeuN + cells↑

Lesion vol↓

No improvement
Lee ST et al. 2005[72]Rat, QAImmortalized NSC (human, HB1.F3)

NeuN + cells↑

Lesion vol↓

Circling behavior↓
Ryu et al. 2004[66]Rat, 3-NPImmortalized NSC (human, HB1.F3)

NeuN + cells↑

Lesion vol↓

Rotarod↓
Roberts et al. 2006[76]Rat, 3-NPNPC (rat)

NPC migration

No change

Beam walking↑
Song et al. 2007[83]Rat, QAESC-derived NSC (human)NeuN + cells↑Circling behavior↓
Aubry et al. 2007[84]Rat, QA

ESC-derived

NSC (human)

DARPP + neuronNot done
Vasey et al. 2010[85]Rat, QAESC-derived NSC (human)

Noggin-primed

NSC migration

Not done

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), known as Lou Gehric disease, is a relentlessly progressive, adult onset neurodegenerative disorder characterized by degeneration and loss of motor neurons in the cerebral cortex, brain stem and spinal cord, leading to muscle wasting and weakness, and eventually to death within 5 years after the onset of its clinical symptoms.[87, 88] The proposed pathogenetic mechanisms of ALS, albeit not fully elucidated, include oxidative stress, protein aggregation, mitochondrial dysfunction, impaired axonal transport, glutamate-mediated excitotoxicity, and insufficient production of neurotrophic factors.[89] To date there is no effective treatment for patients suffering from ALS. Recent studies have indicated that it is possible to generate motor neurons in culture from stem cells that include ESCs and NSCs.[90-93] Mouse ESC-derived motor neurons transplanted into motor neuron-injured rat spinal cord survived and extended axons into the ventral root,[92] and human EGCs transplanted into cerebrospinal fluid of rats with motor neuron injury migrated into the spinal cord and led to improved motor function.[94] Transplantation of NSCs isolated from fetal spinal cord[95] was also effective in delaying disease progression in a mouse ALS model. In a recent study, human spinal cord NSCs derived from an 8-week gestation fetus were transplanted into lumbar spinal cord of superoxide dismutase (SOD)/G93A rats. The results indicated that the neurological function of NSC-transplanted animals was well preserved, but disease onset of transplanted animals was not different from the untreated controls and the overall animal survival was also not affected.[96] A phase I trial of intraspinal injections of fetal-derived NSCs in ALS patients was conducted in the USA. Ten total injections were made into the lumbar spinal cord at a dose of 100 000 cells per injection in 12 ALS patients. Clinical assessments ranging from 6 to 18 months after transplantation demonstrated no evidence of acceleration of disease progression due to the intervention.[97]

A previous study has reported that iPSCs isolated from an ALS patient were differentiated into motor neurons[98] and these patient-derived neurons could be an ideal cellular source for screening new drug candidates. Neurons and glia induced from patient-derived iPSCs are autologous, easily accessible, without immune rejection and with no ethical problem.

The systemic transplantation of NSCs via an intravascular route is probably the least invasive method of cell administration in ALS. Recently rat NSCs labeled with green fluorescent protein were transplanted in a rat ALS model via intravenous tail vein injection and 7 days later 13% of injected cells were found in the motor cortex, hippocmampus and spinal cord. However, no improvement in clinical symptoms was reported.[99]

It is unrealistic to expect the transplantation of stem cells or stem cell-derived motor neurons in ALS patients in a clinical setting will replace lost neurons, integrate into existing neural circuitry and restore motor function. Rather, preventing cell death in host motor neurons via provision of neurotrophic factors by transplanted stem cells or stem cell-derived motor neurons is more realistic and an achievable approach.[100] Recent studies have shown that the application of an adenoviral vector encoding glial cell line-derived growth factor (GDNF) into injured rat facial motor nucleus rescued motor neurons from cell death,[101] and human cortical progenitor cells engineered to express GDNF and transplanted into the spinal cord of ALS rats survived and released the growth factor.[102] Several recent studies have also demonstrated that delivery of vascular endothelial cell growth factor (VEGF) significantly delayed disease onset and prolonged the survival of ALS animal models.[103-105] VEGF is one growth factors that can be used in combination with transplanted stem cells to improve therapeutic efficiency of cellular transplantation. VEGF is an angiogenetic growth factor acting as a potent mitogen and survival factor specific to endothelial cells, and is also known for its neurotrophic and neuroprotective effect against brain injury. Recently we have demonstrated that in a transgenic SOD1/G93A mouse model of ALS[106] intrathecal transplantation of human NSCs over-expressing VEGF induced functional improvement, delayed disease onset for 7 days and extended the survival of animals for 15 days.[107] Immunohistochemical investigation of SOD1/G93A mouse spinal cord demonstrated that the transplanted human NSCs migrated into the spinal cord anterior horn and differentiated into motor neurons.

More recently, we have generated motor neurons from human NSCs and transplanted these cells into the spinal cord of SOD1G93A ALS mouse.[108] Motor neurons were generated by treatment of human NSCs encoding Olig2 basic helix loop helix (bHLH) transcription factor gene (F3.Olig2) with sonic hedgehog (Shh) protein. F3.Olig2-Shh human NSCs expressed motor neuron-specific markers Hb-9, Isl-1 and choline acetyl transferase (ChAT) but did not express cell type-specific markers for oligodendrocytes such as O4, galactocerebroside or CNPase. Control F3.Olig2 NSCs grown in the absence of Shh did not express any of the motor neuron-specific cell type markers. Intrathecal transplantation of motor neuron-committed F3.Olig2-Shh human NSCs into L5 of the spinal cord significantly delayed disease onset (28 days) and prolonged the survival (20 days) of SOD1 G93A ALS mice. Grafted NSCs were found within grey matter and anterior horn of the spinal cord. These results suggest that this treatment modality using genetically modified human NSCs might be of value in the treatment of ALS patients without significant adverse effects.

A summary of preclinical studies of stem cell transplantation in ALS animal models is shown in Table 3.

Table 3. Stem cell-based cell therapy in experimental amyotrophic lateral sclerosis models
ReferenceInjury modelTransplanted cellsAdditional treatmentFunctional outcome
  1. EGC, embryonic germinal stem cell; ESC, embryonic stem cell; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; MN, motor neuron; NPC, neural precursor cell; NSC, neural stem cell; RA, retinoic acid; SHH, sonic hedgehog; SOD, superoxide dismutase; VEGF, vascular endothelial growth factor.
Harper et al. 2004[91]Rat, MN injury-sindivus virusESC (mouse)RA + Shh agonistNot done
Kerr et al. 2003[94]Rat, MN injury-sindivus virusEGC (human)none

BBB-improvement

Limb strength↑

Klein et al. 2005[102]Rat, SOD mutantNPC (human, primary)

GDNF

Gene transfer

BBB-improvement

No survival ext.

Xu et al. 2006[95]Rat, SOD mutantNPC (human, primary)None

BBB-improvement

Extended survival

Hwang et al. 2009[107]Mouse, SOD mutantImmortalized NSC (human HB1.F3)

VEGF

Gene transfer

Rotarod, limb placement↑

Extended survival

Miltrecic et al. 2010[99]Rat, SOD mutantNSC (rat)GFP labeledNot done
Kim KS et al. 2011[108]Mouse, SOD mutantImmortalized NSC (human HB1.F3)

Olig2 Gene transfer

Shh treatment

Rotarod, limb placement↑

Extended survival

Alzheimer's Disease

Alzheimer's disease is characterized by degeneration and loss of neurons and synapses throughout the brain, particularly in the basal forebrain, amygdala, hippocampus and cortical area. Memory and cognitive function of patients progressively decline, patients become demented and prematurely die.[109-111] No effective treatment is currently available except for acetylcholinesterase inhibitors which augment cholinergic function but this is not curative and only a temporary measure.

As for the pathogenesis of AD, the amyloid cascade hypothesis postulates that memory deficits are caused by increased levels of both soluble and insoluble amyloid β (Aβ) peptides, which are derived from the larger amyloid precursor protein (APP) sequential proteolytic processing.[109-111] A recent study has reported that treatment of PDAPP mice, a transgenic mouse model of AD, with anti-Aβ antibody completely restored hippocampal acetylcholine release and high-affinity choline uptake and improved habituation learning.[112] Based on the study, a clinical trial in AD patients is underway in the USA.

Chronically decreasing Aβ levels in the brain has been suggested as a possible therapeutic approach for AD, and experimental evidence indicates that proteinases such as neprilysin,[113] insulin degrading enzyme,[114, 115] plasmin[116] and cathepsin B[117] could be used as therapeutic agents to reduce Aβ levels in AD brain. Recent studies have shown that intracerebral injection of a lentivirus vector expressing human neprilysin in transgenic mouse models of amyloidosis reduced Aβ deposits in the brain and blocked neurodegeneration in the fronal cortex and hippocampus,[118] and that intracerebrally injected fibroblasts over-expressing the human neprilysin gene were found to significantly reduce amyloid plaque burden in the brain of Aβ transgenic mice.[119] These studies support the use of Aβ-degrading proteases as a tool to therapeutically lower Aβ levels and encourage further investigation of ex vivo delivery of protease genes using human NSCs for the treatment of AD. We have recently generated a human NSC line encoding the human neprylysin gene, transplanted these cells into the lateral ventricle of AD transgenic mouse brain, and results are expected shortly.

Ealier studies have indicated that nerve growth factor (NGF) prevents neuronal death and improves memeory in animal models of aging, excitotoxicity and amyloid toxicity,[120-124] and could be used for treating neuronal degeneration and cell death in the AD brain. However, delivery of NGF into the brain is not possible via peripheral administration. Because of its size and polarity, NGF does not cross the blood–brain barrier. In order to overcome this difficulty, a gene therapy approach could be adopted. Using an ex vivo gene therapy approach (genetically modify cells), NGF can be directly inserted into the brain and diffuse for a distance of 2–5 mm.[125] Previously, a phase 1 clinical trial of ex vivo NGF gene delivery was performed in eight mild AD patients, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After a mean follow-up of 22 months in six subjects, long-term adverse effects were not found. Evaluation by Mini-Mental State Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent (ADAS-Cog) suggested improvement in the rate of cognitive decline. Serial positron emission tomography (PET) scans showed significant increases in cortical fluorodeoxyglucose after treatment.[126] Because stem cells can be genetically modified to carry new genes and have high migratory capacity after brain transplantation,[6, 11, 17] they could be used in place of fibroblasts that are known for their immobility following transplantation[43] for delivery of NGF to prevent degeneration of basal forebrain cholinergic neurons. In learning deficit AD model rats induced by okadaic acid injection, transplantation of rat NSCs infected with adenovirus-NGF produced cognitive performance.[127] In a recent study, we used human NSCs in place of rodent NSCs or human fibroblasts to deliver NGF in learning deficit AD model rats. Intrahippocampal injection of ibotenic acid caused severe neuronal loss, resulting in learning and memory deficit.[128] NGF protein released by F3.NGF human NSCs in culture media is 10-fold over the control F3.NSCs at 1.2 μg/106 cells/day. Intra-hippocampal transplantation of F3.NGF cells was found to express NGF and fully improved the learning and memory function of ibotenic learning deficit animals. Transplanted F3.NGF human NSCs were found all over the brain and differentiated into neurons and astrocytes.[128] In another study, brain derived neurotrophic factor (BDNF), a member of the neurotrophin family, secreted by transplanted mouse NSCs was responsible in enhancing cognitive function in triple transgenic AD mice that express pathogenic forms of amyloid precursor protein, presenilin and tau. In these animals cognition was improved without altering Aβ or tau pathology.[129] In other studies in experimental rats with nucleus basalis of Meynert (NBM) lesions induced by ibotenic acid, transplantation of mouse or rat neural precursor cells promoted behavioral recovery.[130, 131]

In AD patients, dysfunction of the presynaptic cholinergic system is one of the causes of cognitive disorders where decreased activity of choline acetyltransferase (ChAT), which is responsible for acetylcholine (ACh) synthesis, is observed.[132] To date, AD therapy has largely been based on small molecules designed to increase ACh concentration by inhibiting acetylcholinesterase.[133] Since therapies with these drugs is only palliative without potential protection against progressive tissue destruction, there is a need for effective therapies for patients with AD, and stem cell-based therapeutic approaches targeting AD should fulfill this requirement. We have recently generated a human NSC line over-expressing human ChAT gene and transplanted these F3.ChAT NSCs into the brain of rat AD models which was generated by intra-hippocampal injection of KA which resulted in severe neuronal loss and profound learning and memory deficit. Intraventricular transplantation of F3.ChAT human NSCs fully restored learning and memory.[134] Similarly F3.ChAT human NSCs were transplanted in AD model rats generated by application of ethylcholine mustard aziridinium ion (AF64A), a cholinergic toxin that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD.[135]Transplantation of F3.ChAT human NSCs in AF64A-treated mice fully restored the learning and memory function of AF64A animals.[136] A recent review article on cell therapy for AD indicated that the stem cell transplant therapy for AD is an extension of the neural stem cells' use in other neurological treatments, such as Parkinson's disease and stroke and could serve as a highly effective therapeutic approach for AD.[137]

A summary of preclinical studies of stem cell-based cell therapy in AD animal models is shown in Table 4.

Table 4. Stem cell-based cell therapy in experimental Alzheimer's disease models
Reference (number)Animal modelTransplanted cellsSpecial featureOutcome
  1. 3XTG, triple transgenic/APP-presenilin-tau; BDNF, brain derived growth factor; ChAT, choline acetyltransferase; ESC, embryonic stem cell; NBM, Nucleus basalis of Meynert; NGF, nerve growth factor; NPC, neural precursor cell; NSC, neural stem cell; SHH, sonic hedgehog protein.
Wang et al. 2006[130]

Mouse

NBM lesion Ibotenic acid –

ESC-derived neurosphere (mouse)ChAT + cells↑Working memory↑
Wu et al. 2008[127]

Rat

Forebrain

Okadaic acid

NSC (rat)

NGF(human)

Gene transfer

Memory↑
Moghadam et al. 2009[131]

Rat

NBM lesion Ibotenic acid

ESC-derived NPC (mouse)Shh-primed

Water maze↑

Spatial probe↑

Blurton-Jones et al. 2009[129]

Mouse

3X TG-AD

NSC (mouse)BDNF-mediated effectWorking memory↑
Park et al. 2012[134]

Rat

Hippocampus

Kainic acid

Immortalized NSC (human, HB1.F3)

ChAT (human)

Gene transfer

Water maze↑

Spatial probe↑

Park et al. 2012[136]

Rat

NBM lesion

AF64A toxin

Immortalized

NSC (human, HB1.F3)

ChAT (human)

Gene transfer

Water maze↑

Spatial probe↑

Lee HJ et al. 2012[128]

Mouse

Hippocampus

Ibotenic acid

Immortalized

NSC (human, HB1.F3)

NGF (human)

Gene transfer

Water maze↑

Spatial probe↑

Perspectives

There are a number of issues to be clarified before adoption of stem cells for cell therapy and gene therapy in clinical medicine, such as which type of stem cells are most suitable for cell replacement therapy in patients with neurological disorders or brain injury, and safety issues related to the risk of tumorigenesis by grafted stem cells. Since neurons could be derived not only from NSCs, but also from ESCs, bone marrow MSCs, adipose tissue-derived MSCs, umbilical cord blood hematopoietic stem cells and even from iPSCs generated from adult somatic cells, the most pressing question is which cells are best suited for cell replacement therapy. Since the presence of NSCs in adult CNS is known, it is only a matter of time before neurons and glial cells are cultured from adult CNS tissue samples. There are ongoing debates as to why oocytes, embryonic or fetal materials should be used to generate stem cells when stem cells could be isolated from adult tissues. However, most research up to now indicates that embryonic or fetal stem cells are significantly more versatile and plastic than adult counterparts.

Previous studies have demonstrated that ESC- or NSC-derived neurons or glial cells could serve as a renewable cell source in cell-based therapy for patients suffering from neurological diseases. However, there exist serious caveats that limit the use of stem cell-derived neurons or glial cells for this purpose. The considerations include: (i) the long-term survival and phenotype stability of stem cell-derived neurons or glial cells in the graft following transplantation are not favorable as earlier studies have demonstrated; (ii) highly purified populations of neuronal cell type derived from ESCs, iPSCs, MSCs or NSCs may contain other neuronal or glial cell types that might produce unpredictable interactions among grafted cells or with host neurons; and (iii) a small number of ESCs, iPSCs, MSCs or NSCs that escaped differentiation and selection processes might expand and form tumors in the graft site following transplantation.

Clonally generated immortalized cell lines of human NSCs as generated by introduction of oncogenes have advantageous features for cell therapy and gene therapy and the features include that human NSCs are homogeneous since they were generated from a single clone, can be expanded to large numbers in vitro, and stable expression of therapeutic genes can be readily achieved. Immortalized human NSCs have emerged as a highly effective source of cells for genetic manipulation and gene transfer into the CNS ex vivo and once transplanted into the damaged brain they survive well, integrate into host tissues and differentiate into both neurons and glial cells. It is known that both extrinsic and inheritable intrinsic signals play important roles in generating cellular diversity in the CNS. By introducing relevant signal molecules or regulatory genes into the human stem cell line, it is now possible to obtain a large number of selected populations of neurons or glial cells from continuously growing human NSCs. Further studies are needed in order to identify the signals for proliferation, differentiation and integration of NSCs and determine favorable conditions of host brain environment for implanted NSCs to survive, prosper and restore the damaged brain.

Acknowledgments

This work was supported by the NRF grants funded by the MEST (2010-0026410 and 2010-0023426) and the Canadian Myelin Research Initiative.

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