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Stem cell based therapies for spinal cord injury
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Review
Stem cell based therapies for spinal cord injury Aikeremujiang Muheremu a,b,c , Jiang Peng b , Qiang Ao c,∗ a
Department of Spine Surgery, Sixth Affiliated Hospital of Xinjiang Medical University, No. 118, Henan West Street, Xinshi District, Urumqi, Xinjiang, China Institute of Orthopaedics, General Hospital of People’s Liberation Army, No. 28 Fuxing Rd, Haidian District, Beijing 100853, China c Department of Tissue Engineering, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning Province 112011, P.R. China b
a r t i c l e
i n f o
Article history: Received 2 February 2016 Received in revised form 25 May 2016 Accepted 28 May 2016 Available online xxx Keywords: Stem cell Transplantation Spinal cord injury
a b s t r a c t Treatment of spinal cord injury has always been a challenge for clinical practitioners and scientists. The development in stem cell based therapies has brought new hopes to patients with spinal cord injuries. In the last a few decades, a variety of stem cells have been used to treat spinal cord injury in animal experiments and some clinical trials. However, there are many technical and ethical challenges to overcome before this novel therapeutic method can be widely applied in clinical practice. With further research in pluripotent stem cells and combined application of genetic and tissue engineering techniques, stem cell based therapies are bond to play increasingly important role in the management of spinal cord injuries. © 2016 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4. 5. 6.
7. 8.
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Possible mechanisms of stem cell based therapies for SCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Replacing the damaged neurons in the spinal cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Protecting the host neurons and preventing apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Promoting axonal regeneration and synapses formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.4. Promoting myelin formation around the remaining and newly grown neural axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The types of stem cells that have been used for the treatment of SCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Neural stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Mesenchymal stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. Embryonic stem cell (ESs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.4. Induced pluripotent stem cells (iPSCs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00 Methods of stem cell transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Time of stem cell transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 New trends in stem cell based therapies for SCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.1. Stem cell therapy combined with gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.2. Stem cell therapy combined with tissue engineering scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.3. Combined use of different types of stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The problems to overcome in the clinical application of stem cells for SCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Background
∗ Corresponding author. E-mail addresses: [email protected] (J. Peng), [email protected] (Q. Ao).
Spinal cord injury (SCI) is one of the most severe complications of spine injuries. 15–40/million people suffer from SCI each year, WHO estimates only 250,000 to 500,000 people suffer SCI each year, including approximately 12,000 cases in the United States. (Mortazavi et al., 2015; One Degree, 2009). The common causes of
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SCI include vehicle accidents, falls, sports and other trauma, infections and tumors. The majority of SCI victims are young patients, who are at the prime of their working age (Tator, 1995). Those injuries not only affect the physical and psychological well-being of those patients, but also bring huge economic burden to their families. In the United States, the social economic lose due to SCI is 8 billion dollars each year. Thus the prevention of SCI and treatment after SCI is an important health care issue. In the current literature, several methods have been described to be effective in the treatment of SCI. However, none of those methods were efficient enough to gain any functional recovery after the injury (Kwon et al., 2010). The major challenge in the treatment of SCI is achieving axonal regeneration and rewiring the spinal cord, which was thought to be impossible until Aguayo et al. (David and Aguayo, 1981) described that central nervous system axons can grow into the peripheral nerve grafts in the early 1980s. Strong proliferation and differentiation potential of stem cells made stem cell transplantation technique possible to replace the injured neurons, modulate the microenvironment, facilitate axonal regeneration and bridge the spinal cord. In this paper, we review the possible mechanisms of stem cell based therapies for SCI, the types of stem cells that can be used for such purpose, the optimal time for stem cell transplantation, different ways to transplant the cells, new trends in stem cell based therapies as well as the problems to solve before using this technique widely in clinical practice. 2. Possible mechanisms of stem cell based therapies for SCI Although it is not absolutely clear, stem cell based therapies for SCI mainly work through several mechanisms.
3. The types of stem cells that have been used for the treatment of SCI A variety of stem cells have been reported to be efficient in the repair of SCI, including neural stem cells, embryonic stem cells, bone marrow mesenchymal stem cells, adipose derived mesenchymal stem cells, umbilical cord blood stem cells, umbilical cord Wharton jelly stem cells, induced pluripotent stem cells, adipose derived mesenchymal stem cells (Table 1). 3.1. Neural stem cells In 1992, Reynolds and Weiss (1992) successfully cultivated neuronal stem cells from mammals by “neurosphere” method, a series of studies since then proved that neural stem cells can be used to promote functional recovery after SCI (Ao et al., 2007; Lu et al., 2014b; Tuszynski et al., 2014). Although this method is easy to carry out and but neural stem cells inside the neurosphere could easily die or differentiate because they could not get enough support from the nutrient composition and the differentiation inhibitory factors in the culture medium. Another well- known method to generate neuronal stem cells is the monolayer method established by Prof Austin Smith in Cambridge Stem Cell Institute (Pollard et al., 2006). This method significantly increases the area of each cell exposed to the culture medium, and provides enough nutritional support to those cells, but requires strict control of the culturing conditions. It has been reported that a combination of those two methods can be applied to harvest large number of neural stem cells with high purity (Dao-Fang et al., 2009). 3.2. Mesenchymal stem cells
2.1. Replacing the damaged neurons in the spinal cord After being transplanted, stem cells can differentiate into neurons and gliocytes, make new connections with the host neurons, and rebuild the neuronal circuit in the spinal cord (Stenudd et al., 2014). 2.2. Protecting the host neurons and preventing apoptosis It has been proven that stem cell transplantation after SCI can down-regulate the expression of genes related to inflammation and apoptosis, and up-regulate the genes with neuron-protective effect, and protect the spinal neurons from secondary changes after the injury (Oliveri et al., 2014). 2.3. Promoting axonal regeneration and synapses formation After transplantation, stem cells interact with the surrounding tissues and produce extracellular matrix as well as several neurotropic factors such as brain derived neurotropic factors, neural growth factor, vascular endothelial growth factor, this changes the microenvironment in the injury site and accelerate the growth of neural axons. The interneurons differentiated from transplanted stem cells can sprout axons and bridge the spinal cord proximal and distal to the site of injury (De Feo et al., 2012; Dalous et al., 2012). 2.4. Promoting myelin formation around the remaining and newly grown neural axons The transplanted stem cells can differentiate into oligodendrocytes and gliocytes, which can promote the formation of myelin and functional recovery in patients with SCI (Cusimano et al., 2012; Yang et al., 2013; Lu et al., 2014a).
Mesenchymal stem cells such as bone marrow stem cells, umbilical cord stem cells and stem cells originated from blood or skin tissues have been applied for the treatment of spinal cord injury (Parr et al., 2007). Cell surface markers can be used to identify meschenchymal stem cells. For example, the typical fractions of mouse MSCs in bone marrow identified by cell sorter are PDGFR␣+/CD51+ fraction (Pinho et al., 2013), and those of human MSCs are Stro-1+/SSEA-4+/CD271+/CD146+ (Bianco, 2014). Although the stem cells supposedly differentiate into neural and glial cells to directly participate in bridging the spinal cord, the current literature is controversial on the ability of mesenchymal stem cells differentiating into neurons (Meletis and Frise ı´n, 2003). However, still most studies evaluating mesenchymal stem cells transplantation for SCI report significantly approved functional recovery in the test subjects. The most likely explanation is that those cells indirectly promote axonal regeneration after SCI by modifying the microenvironment by secreting neurotropic factors and cytokines. The most commonly used mesenchymal stem cells for the treatment of SCI is bone marrow stem cells. Osaka et al. used mesenchymal stem cells derived from bone marrow to treat rat models of contusive spinal cord injury and found that it significantly improved functional outcome (Osaka et al., 2010). Sasaki et al. (2001) found that bone marrow mesenchymal stem cells can be used to repair spinal axons and myelin sheath. Attar et al. (2011) used bone marrow mesenchymal stem cells in clinical trial and none of the patients showed adverse reactions. In the year 2000, Zuk and Safford et al. (Zuk, 2001; Safford et al., 2002) extracted adipose derived mesenchymal stem cells and proved that it can be differentiated into neurons in vivo. Adipose derived mesenchymal stem cells can be easily extracted and cultivated in large numbers, genetically modified and differentiated into neural stem cells. Since the stem cells are extracted from the fat tissue of the patient, there are no ethical concerns regarding its clinical application. However,
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Table 1 Some animal experiments involving stem cell based therapies for spinal cord injuries. Study
Cell sources
SCI models
Functional recovery
Pathologic changes
Karimi-Abdolrezaee et al. (2010)
NPCs extracted from mice forebrain
23 g clip (Walsh) compression injury of rat spinal cord at T6-8 level
NPC transplantation improved locomotor function and did not exacerbate neuropathic pain
Abematsu et al. (2010)
Neuronal stem cells cultivated from mouse forebrains were transplanted with the combination of valporic acid
Mouse SCI model created by Infinite Horizon impactor SCI device at the level of T9-10.
Hind limb function of the animals were dramatically improved
Sasaki et al. (2001)
Bone marrow mesenchymal stem cells obtained from femoral bones in adult LacZ transgenic mice
Not given
Tsuji et al. (2010)
Mouse iPSC-dereived NS/PCs
Kimura et al. (2005)
Mouse neural stem cells differentiated by embryonic stem cells
McDonald et al. (1999)
Neural differentiated mouse embryonic stem cells
Rat SCI model at the level of T10 using a Softex M-150 WZ radiotherapy machine (100 kV, 1.15 mA, SSD 20 cm, dose rate 200 cGy/min) Mice contusive spinal cord injury using an Infinite Horizon Impactor (60 kdyn) at the level of T10 Mice SCI models at T9–T10 level made by a directed impact device, (INP-150; Scholar Tec, Osaka, Japan). Rat spinal cord injury created by NYU impact model at T9-10 level
The transplanted cells were differentiated into oligodendrocytes and facilitated myelin formation around regenerating axons. Transplanted neural stem cells were differentiated into neurons, which connected with endogenous neurons and reconstructed neural circuits. Stem cell transplantation promoted the myelination of regrown axons in the spinal cord
Keirstead et al. (2005)
Oligodendrocytes differentiated from embryonic stem cells
Li et al. (2007)
Neural stem cells and Schwann cells
Lee et al. (2009a)
Human neural stem cell transferred with Bcl-XL gene
Wang et al. (2010)
Bone marrow mesenchymal stem cells planted into chitosan alginate scaffolds
Pritchard et al. (2010)
Biodegradable scaffold implanted with human neural stem cells Human neural stem cells progenitor cells
T9–T10 spinal cord hemisection of African Green Monkey Cervical contusion SCIs in common marmosets using a stereotaxic device
Significant functional recovery was detected in animals after stem cell therapy Bar grip power and spontaneous motor activity recovery was Promoted in SCI primate models
Human iPSC-Derived Neural Stem Cells
Moderate contusive SCI was induced in adult female common marmosets using a modified NYU (New York University) weight-drop device
Functional Recovery was Promoted in SCI primate models after transplantation of hiPSC-NS/PCs
Iwanami et al. (2005)
Kobayashi et al. (2012)
Rat T10 level spinal cord contusion injury was induced using the Infinite Horizon Impactor (Precision Systems, Kentucky, IL) Rat T9 level SCI was induced by aneurysm clip
Rat contusive SCI model at T9 level by a weight dropped from a height of 12.5 mm using an NYU impactor. SD rats were made SCI models by hemi-transecting at T9
there are few reports on the application of adipose derived stem cells in the treatment of SCI. 3.3. Embryonic stem cell (ESs) There are many ways to differentiate ESs into neural precursor cells, neural cells or glial cells (McDonald et al., 1999; Keirstead et al., 2005). There are several studies reporting that transplantation of differentiated ESs can partially recover the hind limb function of
Animals on the experimental group demonstrated better basso mouse scale than PBS control group Animals had better scores motor score, platform hang and rope walk
iPS-SNS–grafted mice had a significantly larger myelinated area at the lesion epicenter than the PBS control mice Myelination of the injured nerve was promoted
Basso–Beattie–Bresnahan Locomotor Rating Scale showed significant improvement after stem cell transplantation Basso, Beattie, Bresnahan Locomotor Rating Scale was significantly improved after stem cell transplantation
Embryonic stem cells were differentiated into glial cells after being transplanted.
No significant difference was found between groups regarding Basso, Beattie, Bresnahan scores Stem cells improved locomotor scores and enhanced accuracy of hindlimb placement in a grid walk Functional recovery was facilitated in SCI rats
NSCs differenting into neurons in a comparatively larger number of mature neurons.
Significant myelin formation around regenerating axons was achieved
Apoptosis in the injury site was significantly reduced
A large number of neurofilament 200 positive fibers and neuron specific enolase positive cells were detected in the lesioned area Contralateral lateral funiculi was preserved from the surgical lesion in all subjects. Grafted human NSPCs survived and differentiated into neurons, astrocytes, and oligodendrocytes; the cavities were smaller than those in sham-operated control animals. Transplantation of hiPSC-NS/PCs enhanced axonal sparing/regrowth and angiogenesis, and prevented the demyelination after SCI compared with that in vehicle control animals
rats after SCI (Nori et al., 2011; Kirkeby et al., 2012). Nori et al. (2011) found that transplantation of human-induced pluripotent stemcell–derived neurospheres into the injured mice spinal cord can significantly promote functional recovery. In 2009, FDA approved the first clinical trial initiated by GeronCorp to use ESs originated oligodendrocyte precursor cells to repair injured spinal cord. After 2 months of immunosuppression therapy, 5 patients included in the study showed no adverse effects related to stem cell transplan-
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Table 2 Some clinical trials involving stem cell based therapy for spinal cord injury. Study
Stem cells
Patients
Results
Attar et al. (2011)
Mesenchymal stem cells
Patients with SCI
Geffner et al. (2008)
Bone marrow mesenchymal stem cells
52 patients with SCI
Kang et al. (2005)
Stem cell transplantation therapy
A patient with spinal cord injury
Syková et al. (2006)
Bone marrow stem cells
Patients with acute and chronic spinal cord injury
Lima et al. (2009)
Olfactory mucosal autografts
Patients with chronic spinal cord injury
Stem Cell Inc
Human Central Nervous System Stem Cells
Patients with thoracic (T2–T11) Spinal Cord
None of the patients showed adverse effects. Stem cell transplantation with different methods have satisfying results with little adverse reactions Achieved satisfying functional and sensorial recovery Majority of patients achieved partial recovery, no adverse reactions were reported Most patients achieved partial functional recovery Not yet revealed
tation. However, GeronCorp withdrew the clinical trial because of the lack of funding in 2011.
3.4. Induced pluripotent stem cells (iPSCs) In 2006, Takahashi and Yamanaka (2006) first successfully induced mice fibroblasts into dedifferentiated cells with similar characteristics with embryonic stem cells. Since then several authors reported that iPSCs can be differentiated into neural cells and glial cells and applied in the treatment of mice, rat and monkey spinal cord injury models (Tsuji et al., 2010; Kimura et al., 2005; Attar et al., 2011). Puri and Nagy (2012) compared ESs and iPSCs and found that they have similar morphological, functional and genetic characteristics. Considering that the application of iPSCs has no ethical obstacles, it could be a candidate with great potential.
4. Methods of stem cell transplantation Stem cells can be transplanted via different routes such as intramedullary, intrathecal, intraventricular and intravascular routes. Among those methods, intramedullary methods is the most effective but at the same time most invasive method, while intravascular method is the least effective and yet least invasive. Geffner et al. (2008) used intrathecal and intravascular routes to transplant bone marrow mesenchymal stem cells into 52 patients with SCI. All those patients achieved partial recovery with no serious adverse outcomes from the transplantation. Regional transplantation can achieve the highest concentration of stem cells in the injury site, where they are most needed. However, this method often requires a second surgery, which increases the costs of treatment and chances of infection. Neuhuber et al. (2008) reported that intrathecal transplantation via lumber puncture using large amount of stem cells can be considered to replace direct regional transplantation as an effective and less invasive method. Lee et al. (2009b) reported that percutaneous transplantation is a method which requires easily manageable technique and causes little damage to the normal tissues. However, in the study of Takahashi et al. (2011), neural stem/progenitor cells were administered via different routes to treat SCI in mice. In this study, intrathecal transplantation method resulted in less survival rate of transplanted cells and no functional recovery on the animals, and intravenous single cell injection of mouse NSPCs resulted in fatal pulmonary thromboembolism. In spite of numerous studies on different methods of stem cell engraftment, the type of animal models, injury mechanisms and the variety of stem cells are different and not comparable among the studies. Moreover, most of those studies were carried out in animal models, and the conclusions from these studies cannot be directly applied for human subjects. Further study is
needed to find the best route for stem cell transplantation to treat patients with SCI. 5. Time of stem cell transplantation Appropriate timing of transplantation is essential for the survival of the transplanted cells, and the regeneration in the injured nervous system (Nishimura et al., 2013). However, there is no consensus about the appropriate timing of transplantation after SCI. Some authors believe that the tremendous amount of neurotoxins generated after SCI creates a hostile environment for transplanted cells to survive, proliferate and differentiate, hence it is better to undergo stem cell transplantation one or two weeks after SCI (Antonic et al., 2013; Sabelström et al., 2013). Some other authors believe that stem cell transplantation should be carried out immediately after SCI, because the inflammatory reaction after SCI has little effect on the transplanted stem cells, and those stem cells may be able to alter the microenvironment at the injury site, prevent the apoptosis of host neural cells, and reduce the secondary damage (Nakajima et al., 2012). Li et al. (2010) reported that transplanting stem cells multiple times after the injury has better effect that transplanting just once, and they believe that the optimal frequency of stem cell transplantation after SCI is three times. However, similar to the reason described above, the types of stem cells, animal models and transplantation methods are different in different studies, the current literature does not have adequate information to draw a conclusion on the best timing of stem cell transplantation. 6. New trends in stem cell based therapies for SCI In the last decade, with the application of gene and tissue engineering techniques, stem cell transplantation treatments for SCI has become increasingly effective. 6.1. Stem cell therapy combined with gene therapy The basic strategy of gene therapy is to transfer some specific genes with curative effect to the site of injury and create favorable microenvironment for the repair of spinal cord. Genetically modified stem cells can not only replace the damaged neural cells, but also overexpress specific neurotropic factors to rebuild the neural circuit in the spinal cord (Kumagai et al., 2013; Castellanos et al., 2002; Lin et al., 2013). It is possible that after figuring out the genetic characteristics of some animals that can achieve complete spinal cord regeneration after injury, zebra fish for example, the genes necessary for neural regeneration after SCI can be transferred to the injury site to promote the axonal regeneration and functional recovery. However, the viruses utilized to transduce some genes are often integrated in the vicinity of gene promoters, creating a risk
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of altering the expression pattern of nearby endogenous genes and resulting in tumor formation. For example, in the study of HaceinBey-Abina et al. (2003), leukemia was developed in 2 of 10 patients with X-linked severe combined immunodeficiency who underwent gene therapy with a retrovirus vector. Thus safety of genetically engineered stem cells should be guaranteed by extensive animal studies before such therapies can be used in clinical trials. 6.2. Stem cell therapy combined with tissue engineering scaffolds Tissue engineered scaffolds with adequate histocompatibility could be used to bridge the defect formed by cavity and glial scar tissues, and promote axonal regeneration by improving the microenvironment. In their most recent publication, Tuszynski et al. (Kadoya et al., 2016) reported the robust regeneration of cortico-spinal tract after homologous neural grafts transplantation. However, the perfect combination of tissue engineering materials and stem cells cannot be easily achieved. A lot more research should be done to fabricate ideal scaffold from which the transplanted stem cells can migrate into the host spinal cord tissue while the neural cells and axons from the host tissue can grow into the scaffold. 6.3. Combined use of different types of stem cells There are many studies advocating that a combination of different types of stem cells can be more effective than a single cell to repair spinal cord when transplanted after SCI (Tetzlaff et al., 2011; Ruff et al., 2012). It is possible that different types of cells could secrete different neurotropic factors and promote axonal regeneration in the injured region. There is still much to explore the working mechanism of such method and find an optimal combination of types, quantity and density of different stem cells to achieve best functional recovery after SCI. 7. The problems to overcome in the clinical application of stem cells for SCI In the last two decades, with the advancements in cellular, genetic and tissue engineering techniques, and with the exploration of molecular mechanisms of the pathological changes after SCI, encouraging results have been achieved in some animal and clinical studies (Tables 1,2). However, before this technique can be used in the clinical practice, some issues still need to addressed. First of all, the limited sources and ethical challenges concerning neural stem cells and ESs greatly hindered their clinical application. Although bone marrow mesenchymal stem cells have no such problems, they need 2–4 weeks in-vitro culturing before they can be used for transplantation. The applications of iPSCs may effectively solve those problems, but years of animal research is needed before they can be used in the clinical trials (RaJC et al., 2011). Moreover, because there are significant differences between patients with SCI regarding the degree and site of the injury, it is hard to find enough number of patients with similar basic characteristics to carry out randomized controlled trials (Mackay-Sim and Féron, 2013), making it almost impossible to avoid patient selection bias. Preventing tumor formation and immune rejection is another major issue to overcome for successful stem cell transplantation (Okano et al., 2013). A failsafe system after stem cell transplantation for SCI is mandatory in clinical trials. Patients should be followed by routine outpatient visits and any possible tumor formation should be detected, confirmed and eliminated at an early stage. Amariglio et al. reported a patient who received donor-cell (NSPCs)-derived neural stem cell transplantation and developed brain tumor (Amariglio et al., 2009). Dlouhy et al. reported a case with autograft-derived spinal
5
cord mass after olfactory mucosal cell transplantation. In both cases timely surgical resection avoided fatal consequences (Dlouhy et al., 2014). 8. Conclusions Stem cell based therapies have provided us new methods to restore function in patients with SCI. A variety of stem cells with different transplantation techniques have been used in animal studies and a few clinical trials. However, functional recovery under the injury level after complete SCI is still not possible. With the exploration of molecular mechanisms of neural regeneration after SCI in animal models and clinical settings, and with the application of genetic and tissue engineering techniques, stem cell based therapies will surely help us achieve better recovery after SCI in the near future. Funding This research was funded by The Natural Science Foundation of China (NO: 81360194); the National High Technology Research and Development Program of China (“863”Program, No. 2012AA020905); and the National Basic Research Program of China (973 program, No. 2014CB542200). References Abematsu, M., Tsujimura, K., Yamano, M., Saito, M., Kohno, K., Kohyama, J., Nakashima, K., 2010. Neurons derived from transplanted neural stem cells restore disrupted neuronal circuitry in a mouse model of spinal cord injury. J. Clin. Invest. 120 (9), 3255. Amariglio, N., Hirshberg, A., Scheithauer, B.W., Cohen, Y., Loewenthal, R., Trakhtenbrot, L., Paz, N., Koren-Michowitz, M., Waldman, D., Leider-Trejo, L., Toren, A., Constantini, S., Rechavi, G., 2009. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 6 (2) (e1000029). Antonic, A., Sena, E.S., Lees, J.S., Wills, T.E., Skeers, P., Batchelor, P.E., Howells, D.W., 2013. Stem cell transplantation in traumatic spinal cord injury: a systematic review and meta-analysis of animal studies. PLoS Biol. 11 (12), e1001738. Ao, Q., Wang, A.J., Chen, G.Q., et al., 2007. Combined transplantation of neural stem cells and olfactory unsheathing cells for the repair of spinal cord injuries. Med. Hypotheses 69 (6), 1234–1237. Attar, A., Ayten, M., Ozdemir, M., Ozgencil, E., Bozkurt, M., Kaptanoglu, E., Kanpolat, Y., 2011. An attempt to treat patients who have injured spinal cords with intralesional implantation of concentrated autologous bone marrow cells. Cytotherapy 13 (1), 54–60. Bianco, P., 2014. Mesenchymal stem cells. Annu. Rev. Cell Dev. Biol. 30, 677–704. Castellanos, D.A., Tsoulfas, P., Frydel, B.R., et al., 2002. Over expression enhances survival and migration of neural stem cell transplants in the rat spinal cord. Cell Transplant. 11 (3), 297–307. Cusimano, M., Biziato, D., Brambilla, E., Donegà, M., Alfaro-Cervello, C., Snider, S., 2012. Transplanted neural stem/precursor cells instruct phagocytes and reduce secondary tissue damage in the injured spinal cord. Brain 135 (2), 447–460. Dalous, J., Larghero, J., Baud, O., 2012. Transplantation of umbilical cord-derived mesenchymal stem cells as a novel strategy to protect the central nervous system: technical aspects, preclinical studies, and clinical perspectives. Pediatr. Res. 71 (2–4), 482–490. Dao-Fang, D., San-Li, X., Ming-Ming, Z., 2009. The monolayer culturing of the neural stem cell clone and its qualification. Prog. Biochem. Biophys. 36 (1), 72–76. David, S., Aguayo, A.J., 1981. Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats. Science 214, 931–933. De Feo, D., Merlini, A., Laterza, C., Martino, G., 2012. Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection. Curr. Opin. Neurol. 25 (3), 322–333. Dlouhy, B.J., Awe, O., Rao, R.C., Kirby, P.A., Hitchon, P.W., 2014. Autograft-derived spinal cord mass following olfactory mucosal cell transplantation in a spinal cord injury patient: case report. J. Neurosurg. Spine 21 (4), 618–622. Geffner, L.F., Santacruz, P., Izurieta, M., Flor, L., Maldonado, B., Auad, A.H., Silva, F., 2008. Administration of autologous bone marrow stem cells into spinal cord injury patients via multiple routes is safe and improves their quality of life: comprehensive case studies. Cell Transplant. 17 (12), 1277–1293. Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., et al., 2003. LMO2- associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419. Iwanami, A., Kaneko, S., Nakamura, M., Kanemura, Y., Mori, H., Kobayashi, S., Yamasaki, M., Morishima, S., Ishii, H., Ando, K., Tanioka, Y., Tamaoki, N.,
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Review
Stem cell based therapies for spinal cord injury Aikeremujiang Muheremu a,b,c , Jiang Peng b , Qiang Ao c,∗ a
Department of Spine Surgery, Sixth Affiliated Hospital of Xinjiang Medical University, No. 118, Henan West Street, Xinshi District, Urumqi, Xinjiang, China Institute of Orthopaedics, General Hospital of People’s Liberation Army, No. 28 Fuxing Rd, Haidian District, Beijing 100853, China c Department of Tissue Engineering, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning Province 112011, P.R. China b
a r t i c l e
i n f o
Article history: Received 2 February 2016 Received in revised form 25 May 2016 Accepted 28 May 2016 Available online xxx Keywords: Stem cell Transplantation Spinal cord injury
a b s t r a c t Treatment of spinal cord injury has always been a challenge for clinical practitioners and scientists. The development in stem cell based therapies has brought new hopes to patients with spinal cord injuries. In the last a few decades, a variety of stem cells have been used to treat spinal cord injury in animal experiments and some clinical trials. However, there are many technical and ethical challenges to overcome before this novel therapeutic method can be widely applied in clinical practice. With further research in pluripotent stem cells and combined application of genetic and tissue engineering techniques, stem cell based therapies are bond to play increasingly important role in the management of spinal cord injuries. © 2016 Elsevier Ltd. All rights reserved.
Contents 1. 2.
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Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Possible mechanisms of stem cell based therapies for SCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Replacing the damaged neurons in the spinal cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Protecting the host neurons and preventing apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Promoting axonal regeneration and synapses formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.4. Promoting myelin formation around the remaining and newly grown neural axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The types of stem cells that have been used for the treatment of SCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Neural stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Mesenchymal stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. Embryonic stem cell (ESs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.4. Induced pluripotent stem cells (iPSCs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00 Methods of stem cell transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Time of stem cell transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 New trends in stem cell based therapies for SCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.1. Stem cell therapy combined with gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.2. Stem cell therapy combined with tissue engineering scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.3. Combined use of different types of stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The problems to overcome in the clinical application of stem cells for SCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Background
∗ Corresponding author. E-mail addresses: [email protected] (J. Peng), [email protected] (Q. Ao).
Spinal cord injury (SCI) is one of the most severe complications of spine injuries. 15–40/million people suffer from SCI each year, WHO estimates only 250,000 to 500,000 people suffer SCI each year, including approximately 12,000 cases in the United States. (Mortazavi et al., 2015; One Degree, 2009). The common causes of
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SCI include vehicle accidents, falls, sports and other trauma, infections and tumors. The majority of SCI victims are young patients, who are at the prime of their working age (Tator, 1995). Those injuries not only affect the physical and psychological well-being of those patients, but also bring huge economic burden to their families. In the United States, the social economic lose due to SCI is 8 billion dollars each year. Thus the prevention of SCI and treatment after SCI is an important health care issue. In the current literature, several methods have been described to be effective in the treatment of SCI. However, none of those methods were efficient enough to gain any functional recovery after the injury (Kwon et al., 2010). The major challenge in the treatment of SCI is achieving axonal regeneration and rewiring the spinal cord, which was thought to be impossible until Aguayo et al. (David and Aguayo, 1981) described that central nervous system axons can grow into the peripheral nerve grafts in the early 1980s. Strong proliferation and differentiation potential of stem cells made stem cell transplantation technique possible to replace the injured neurons, modulate the microenvironment, facilitate axonal regeneration and bridge the spinal cord. In this paper, we review the possible mechanisms of stem cell based therapies for SCI, the types of stem cells that can be used for such purpose, the optimal time for stem cell transplantation, different ways to transplant the cells, new trends in stem cell based therapies as well as the problems to solve before using this technique widely in clinical practice. 2. Possible mechanisms of stem cell based therapies for SCI Although it is not absolutely clear, stem cell based therapies for SCI mainly work through several mechanisms.
3. The types of stem cells that have been used for the treatment of SCI A variety of stem cells have been reported to be efficient in the repair of SCI, including neural stem cells, embryonic stem cells, bone marrow mesenchymal stem cells, adipose derived mesenchymal stem cells, umbilical cord blood stem cells, umbilical cord Wharton jelly stem cells, induced pluripotent stem cells, adipose derived mesenchymal stem cells (Table 1). 3.1. Neural stem cells In 1992, Reynolds and Weiss (1992) successfully cultivated neuronal stem cells from mammals by “neurosphere” method, a series of studies since then proved that neural stem cells can be used to promote functional recovery after SCI (Ao et al., 2007; Lu et al., 2014b; Tuszynski et al., 2014). Although this method is easy to carry out and but neural stem cells inside the neurosphere could easily die or differentiate because they could not get enough support from the nutrient composition and the differentiation inhibitory factors in the culture medium. Another well- known method to generate neuronal stem cells is the monolayer method established by Prof Austin Smith in Cambridge Stem Cell Institute (Pollard et al., 2006). This method significantly increases the area of each cell exposed to the culture medium, and provides enough nutritional support to those cells, but requires strict control of the culturing conditions. It has been reported that a combination of those two methods can be applied to harvest large number of neural stem cells with high purity (Dao-Fang et al., 2009). 3.2. Mesenchymal stem cells
2.1. Replacing the damaged neurons in the spinal cord After being transplanted, stem cells can differentiate into neurons and gliocytes, make new connections with the host neurons, and rebuild the neuronal circuit in the spinal cord (Stenudd et al., 2014). 2.2. Protecting the host neurons and preventing apoptosis It has been proven that stem cell transplantation after SCI can down-regulate the expression of genes related to inflammation and apoptosis, and up-regulate the genes with neuron-protective effect, and protect the spinal neurons from secondary changes after the injury (Oliveri et al., 2014). 2.3. Promoting axonal regeneration and synapses formation After transplantation, stem cells interact with the surrounding tissues and produce extracellular matrix as well as several neurotropic factors such as brain derived neurotropic factors, neural growth factor, vascular endothelial growth factor, this changes the microenvironment in the injury site and accelerate the growth of neural axons. The interneurons differentiated from transplanted stem cells can sprout axons and bridge the spinal cord proximal and distal to the site of injury (De Feo et al., 2012; Dalous et al., 2012). 2.4. Promoting myelin formation around the remaining and newly grown neural axons The transplanted stem cells can differentiate into oligodendrocytes and gliocytes, which can promote the formation of myelin and functional recovery in patients with SCI (Cusimano et al., 2012; Yang et al., 2013; Lu et al., 2014a).
Mesenchymal stem cells such as bone marrow stem cells, umbilical cord stem cells and stem cells originated from blood or skin tissues have been applied for the treatment of spinal cord injury (Parr et al., 2007). Cell surface markers can be used to identify meschenchymal stem cells. For example, the typical fractions of mouse MSCs in bone marrow identified by cell sorter are PDGFR␣+/CD51+ fraction (Pinho et al., 2013), and those of human MSCs are Stro-1+/SSEA-4+/CD271+/CD146+ (Bianco, 2014). Although the stem cells supposedly differentiate into neural and glial cells to directly participate in bridging the spinal cord, the current literature is controversial on the ability of mesenchymal stem cells differentiating into neurons (Meletis and Frise ı´n, 2003). However, still most studies evaluating mesenchymal stem cells transplantation for SCI report significantly approved functional recovery in the test subjects. The most likely explanation is that those cells indirectly promote axonal regeneration after SCI by modifying the microenvironment by secreting neurotropic factors and cytokines. The most commonly used mesenchymal stem cells for the treatment of SCI is bone marrow stem cells. Osaka et al. used mesenchymal stem cells derived from bone marrow to treat rat models of contusive spinal cord injury and found that it significantly improved functional outcome (Osaka et al., 2010). Sasaki et al. (2001) found that bone marrow mesenchymal stem cells can be used to repair spinal axons and myelin sheath. Attar et al. (2011) used bone marrow mesenchymal stem cells in clinical trial and none of the patients showed adverse reactions. In the year 2000, Zuk and Safford et al. (Zuk, 2001; Safford et al., 2002) extracted adipose derived mesenchymal stem cells and proved that it can be differentiated into neurons in vivo. Adipose derived mesenchymal stem cells can be easily extracted and cultivated in large numbers, genetically modified and differentiated into neural stem cells. Since the stem cells are extracted from the fat tissue of the patient, there are no ethical concerns regarding its clinical application. However,
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Table 1 Some animal experiments involving stem cell based therapies for spinal cord injuries. Study
Cell sources
SCI models
Functional recovery
Pathologic changes
Karimi-Abdolrezaee et al. (2010)
NPCs extracted from mice forebrain
23 g clip (Walsh) compression injury of rat spinal cord at T6-8 level
NPC transplantation improved locomotor function and did not exacerbate neuropathic pain
Abematsu et al. (2010)
Neuronal stem cells cultivated from mouse forebrains were transplanted with the combination of valporic acid
Mouse SCI model created by Infinite Horizon impactor SCI device at the level of T9-10.
Hind limb function of the animals were dramatically improved
Sasaki et al. (2001)
Bone marrow mesenchymal stem cells obtained from femoral bones in adult LacZ transgenic mice
Not given
Tsuji et al. (2010)
Mouse iPSC-dereived NS/PCs
Kimura et al. (2005)
Mouse neural stem cells differentiated by embryonic stem cells
McDonald et al. (1999)
Neural differentiated mouse embryonic stem cells
Rat SCI model at the level of T10 using a Softex M-150 WZ radiotherapy machine (100 kV, 1.15 mA, SSD 20 cm, dose rate 200 cGy/min) Mice contusive spinal cord injury using an Infinite Horizon Impactor (60 kdyn) at the level of T10 Mice SCI models at T9–T10 level made by a directed impact device, (INP-150; Scholar Tec, Osaka, Japan). Rat spinal cord injury created by NYU impact model at T9-10 level
The transplanted cells were differentiated into oligodendrocytes and facilitated myelin formation around regenerating axons. Transplanted neural stem cells were differentiated into neurons, which connected with endogenous neurons and reconstructed neural circuits. Stem cell transplantation promoted the myelination of regrown axons in the spinal cord
Keirstead et al. (2005)
Oligodendrocytes differentiated from embryonic stem cells
Li et al. (2007)
Neural stem cells and Schwann cells
Lee et al. (2009a)
Human neural stem cell transferred with Bcl-XL gene
Wang et al. (2010)
Bone marrow mesenchymal stem cells planted into chitosan alginate scaffolds
Pritchard et al. (2010)
Biodegradable scaffold implanted with human neural stem cells Human neural stem cells progenitor cells
T9–T10 spinal cord hemisection of African Green Monkey Cervical contusion SCIs in common marmosets using a stereotaxic device
Significant functional recovery was detected in animals after stem cell therapy Bar grip power and spontaneous motor activity recovery was Promoted in SCI primate models
Human iPSC-Derived Neural Stem Cells
Moderate contusive SCI was induced in adult female common marmosets using a modified NYU (New York University) weight-drop device
Functional Recovery was Promoted in SCI primate models after transplantation of hiPSC-NS/PCs
Iwanami et al. (2005)
Kobayashi et al. (2012)
Rat T10 level spinal cord contusion injury was induced using the Infinite Horizon Impactor (Precision Systems, Kentucky, IL) Rat T9 level SCI was induced by aneurysm clip
Rat contusive SCI model at T9 level by a weight dropped from a height of 12.5 mm using an NYU impactor. SD rats were made SCI models by hemi-transecting at T9
there are few reports on the application of adipose derived stem cells in the treatment of SCI. 3.3. Embryonic stem cell (ESs) There are many ways to differentiate ESs into neural precursor cells, neural cells or glial cells (McDonald et al., 1999; Keirstead et al., 2005). There are several studies reporting that transplantation of differentiated ESs can partially recover the hind limb function of
Animals on the experimental group demonstrated better basso mouse scale than PBS control group Animals had better scores motor score, platform hang and rope walk
iPS-SNS–grafted mice had a significantly larger myelinated area at the lesion epicenter than the PBS control mice Myelination of the injured nerve was promoted
Basso–Beattie–Bresnahan Locomotor Rating Scale showed significant improvement after stem cell transplantation Basso, Beattie, Bresnahan Locomotor Rating Scale was significantly improved after stem cell transplantation
Embryonic stem cells were differentiated into glial cells after being transplanted.
No significant difference was found between groups regarding Basso, Beattie, Bresnahan scores Stem cells improved locomotor scores and enhanced accuracy of hindlimb placement in a grid walk Functional recovery was facilitated in SCI rats
NSCs differenting into neurons in a comparatively larger number of mature neurons.
Significant myelin formation around regenerating axons was achieved
Apoptosis in the injury site was significantly reduced
A large number of neurofilament 200 positive fibers and neuron specific enolase positive cells were detected in the lesioned area Contralateral lateral funiculi was preserved from the surgical lesion in all subjects. Grafted human NSPCs survived and differentiated into neurons, astrocytes, and oligodendrocytes; the cavities were smaller than those in sham-operated control animals. Transplantation of hiPSC-NS/PCs enhanced axonal sparing/regrowth and angiogenesis, and prevented the demyelination after SCI compared with that in vehicle control animals
rats after SCI (Nori et al., 2011; Kirkeby et al., 2012). Nori et al. (2011) found that transplantation of human-induced pluripotent stemcell–derived neurospheres into the injured mice spinal cord can significantly promote functional recovery. In 2009, FDA approved the first clinical trial initiated by GeronCorp to use ESs originated oligodendrocyte precursor cells to repair injured spinal cord. After 2 months of immunosuppression therapy, 5 patients included in the study showed no adverse effects related to stem cell transplan-
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Table 2 Some clinical trials involving stem cell based therapy for spinal cord injury. Study
Stem cells
Patients
Results
Attar et al. (2011)
Mesenchymal stem cells
Patients with SCI
Geffner et al. (2008)
Bone marrow mesenchymal stem cells
52 patients with SCI
Kang et al. (2005)
Stem cell transplantation therapy
A patient with spinal cord injury
Syková et al. (2006)
Bone marrow stem cells
Patients with acute and chronic spinal cord injury
Lima et al. (2009)
Olfactory mucosal autografts
Patients with chronic spinal cord injury
Stem Cell Inc
Human Central Nervous System Stem Cells
Patients with thoracic (T2–T11) Spinal Cord
None of the patients showed adverse effects. Stem cell transplantation with different methods have satisfying results with little adverse reactions Achieved satisfying functional and sensorial recovery Majority of patients achieved partial recovery, no adverse reactions were reported Most patients achieved partial functional recovery Not yet revealed
tation. However, GeronCorp withdrew the clinical trial because of the lack of funding in 2011.
3.4. Induced pluripotent stem cells (iPSCs) In 2006, Takahashi and Yamanaka (2006) first successfully induced mice fibroblasts into dedifferentiated cells with similar characteristics with embryonic stem cells. Since then several authors reported that iPSCs can be differentiated into neural cells and glial cells and applied in the treatment of mice, rat and monkey spinal cord injury models (Tsuji et al., 2010; Kimura et al., 2005; Attar et al., 2011). Puri and Nagy (2012) compared ESs and iPSCs and found that they have similar morphological, functional and genetic characteristics. Considering that the application of iPSCs has no ethical obstacles, it could be a candidate with great potential.
4. Methods of stem cell transplantation Stem cells can be transplanted via different routes such as intramedullary, intrathecal, intraventricular and intravascular routes. Among those methods, intramedullary methods is the most effective but at the same time most invasive method, while intravascular method is the least effective and yet least invasive. Geffner et al. (2008) used intrathecal and intravascular routes to transplant bone marrow mesenchymal stem cells into 52 patients with SCI. All those patients achieved partial recovery with no serious adverse outcomes from the transplantation. Regional transplantation can achieve the highest concentration of stem cells in the injury site, where they are most needed. However, this method often requires a second surgery, which increases the costs of treatment and chances of infection. Neuhuber et al. (2008) reported that intrathecal transplantation via lumber puncture using large amount of stem cells can be considered to replace direct regional transplantation as an effective and less invasive method. Lee et al. (2009b) reported that percutaneous transplantation is a method which requires easily manageable technique and causes little damage to the normal tissues. However, in the study of Takahashi et al. (2011), neural stem/progenitor cells were administered via different routes to treat SCI in mice. In this study, intrathecal transplantation method resulted in less survival rate of transplanted cells and no functional recovery on the animals, and intravenous single cell injection of mouse NSPCs resulted in fatal pulmonary thromboembolism. In spite of numerous studies on different methods of stem cell engraftment, the type of animal models, injury mechanisms and the variety of stem cells are different and not comparable among the studies. Moreover, most of those studies were carried out in animal models, and the conclusions from these studies cannot be directly applied for human subjects. Further study is
needed to find the best route for stem cell transplantation to treat patients with SCI. 5. Time of stem cell transplantation Appropriate timing of transplantation is essential for the survival of the transplanted cells, and the regeneration in the injured nervous system (Nishimura et al., 2013). However, there is no consensus about the appropriate timing of transplantation after SCI. Some authors believe that the tremendous amount of neurotoxins generated after SCI creates a hostile environment for transplanted cells to survive, proliferate and differentiate, hence it is better to undergo stem cell transplantation one or two weeks after SCI (Antonic et al., 2013; Sabelström et al., 2013). Some other authors believe that stem cell transplantation should be carried out immediately after SCI, because the inflammatory reaction after SCI has little effect on the transplanted stem cells, and those stem cells may be able to alter the microenvironment at the injury site, prevent the apoptosis of host neural cells, and reduce the secondary damage (Nakajima et al., 2012). Li et al. (2010) reported that transplanting stem cells multiple times after the injury has better effect that transplanting just once, and they believe that the optimal frequency of stem cell transplantation after SCI is three times. However, similar to the reason described above, the types of stem cells, animal models and transplantation methods are different in different studies, the current literature does not have adequate information to draw a conclusion on the best timing of stem cell transplantation. 6. New trends in stem cell based therapies for SCI In the last decade, with the application of gene and tissue engineering techniques, stem cell transplantation treatments for SCI has become increasingly effective. 6.1. Stem cell therapy combined with gene therapy The basic strategy of gene therapy is to transfer some specific genes with curative effect to the site of injury and create favorable microenvironment for the repair of spinal cord. Genetically modified stem cells can not only replace the damaged neural cells, but also overexpress specific neurotropic factors to rebuild the neural circuit in the spinal cord (Kumagai et al., 2013; Castellanos et al., 2002; Lin et al., 2013). It is possible that after figuring out the genetic characteristics of some animals that can achieve complete spinal cord regeneration after injury, zebra fish for example, the genes necessary for neural regeneration after SCI can be transferred to the injury site to promote the axonal regeneration and functional recovery. However, the viruses utilized to transduce some genes are often integrated in the vicinity of gene promoters, creating a risk
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of altering the expression pattern of nearby endogenous genes and resulting in tumor formation. For example, in the study of HaceinBey-Abina et al. (2003), leukemia was developed in 2 of 10 patients with X-linked severe combined immunodeficiency who underwent gene therapy with a retrovirus vector. Thus safety of genetically engineered stem cells should be guaranteed by extensive animal studies before such therapies can be used in clinical trials. 6.2. Stem cell therapy combined with tissue engineering scaffolds Tissue engineered scaffolds with adequate histocompatibility could be used to bridge the defect formed by cavity and glial scar tissues, and promote axonal regeneration by improving the microenvironment. In their most recent publication, Tuszynski et al. (Kadoya et al., 2016) reported the robust regeneration of cortico-spinal tract after homologous neural grafts transplantation. However, the perfect combination of tissue engineering materials and stem cells cannot be easily achieved. A lot more research should be done to fabricate ideal scaffold from which the transplanted stem cells can migrate into the host spinal cord tissue while the neural cells and axons from the host tissue can grow into the scaffold. 6.3. Combined use of different types of stem cells There are many studies advocating that a combination of different types of stem cells can be more effective than a single cell to repair spinal cord when transplanted after SCI (Tetzlaff et al., 2011; Ruff et al., 2012). It is possible that different types of cells could secrete different neurotropic factors and promote axonal regeneration in the injured region. There is still much to explore the working mechanism of such method and find an optimal combination of types, quantity and density of different stem cells to achieve best functional recovery after SCI. 7. The problems to overcome in the clinical application of stem cells for SCI In the last two decades, with the advancements in cellular, genetic and tissue engineering techniques, and with the exploration of molecular mechanisms of the pathological changes after SCI, encouraging results have been achieved in some animal and clinical studies (Tables 1,2). However, before this technique can be used in the clinical practice, some issues still need to addressed. First of all, the limited sources and ethical challenges concerning neural stem cells and ESs greatly hindered their clinical application. Although bone marrow mesenchymal stem cells have no such problems, they need 2–4 weeks in-vitro culturing before they can be used for transplantation. The applications of iPSCs may effectively solve those problems, but years of animal research is needed before they can be used in the clinical trials (RaJC et al., 2011). Moreover, because there are significant differences between patients with SCI regarding the degree and site of the injury, it is hard to find enough number of patients with similar basic characteristics to carry out randomized controlled trials (Mackay-Sim and Féron, 2013), making it almost impossible to avoid patient selection bias. Preventing tumor formation and immune rejection is another major issue to overcome for successful stem cell transplantation (Okano et al., 2013). A failsafe system after stem cell transplantation for SCI is mandatory in clinical trials. Patients should be followed by routine outpatient visits and any possible tumor formation should be detected, confirmed and eliminated at an early stage. Amariglio et al. reported a patient who received donor-cell (NSPCs)-derived neural stem cell transplantation and developed brain tumor (Amariglio et al., 2009). Dlouhy et al. reported a case with autograft-derived spinal
5
cord mass after olfactory mucosal cell transplantation. In both cases timely surgical resection avoided fatal consequences (Dlouhy et al., 2014). 8. Conclusions Stem cell based therapies have provided us new methods to restore function in patients with SCI. A variety of stem cells with different transplantation techniques have been used in animal studies and a few clinical trials. However, functional recovery under the injury level after complete SCI is still not possible. With the exploration of molecular mechanisms of neural regeneration after SCI in animal models and clinical settings, and with the application of genetic and tissue engineering techniques, stem cell based therapies will surely help us achieve better recovery after SCI in the near future. Funding This research was funded by The Natural Science Foundation of China (NO: 81360194); the National High Technology Research and Development Program of China (“863”Program, No. 2012AA020905); and the National Basic Research Program of China (973 program, No. 2014CB542200). References Abematsu, M., Tsujimura, K., Yamano, M., Saito, M., Kohno, K., Kohyama, J., Nakashima, K., 2010. Neurons derived from transplanted neural stem cells restore disrupted neuronal circuitry in a mouse model of spinal cord injury. J. Clin. Invest. 120 (9), 3255. Amariglio, N., Hirshberg, A., Scheithauer, B.W., Cohen, Y., Loewenthal, R., Trakhtenbrot, L., Paz, N., Koren-Michowitz, M., Waldman, D., Leider-Trejo, L., Toren, A., Constantini, S., Rechavi, G., 2009. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 6 (2) (e1000029). Antonic, A., Sena, E.S., Lees, J.S., Wills, T.E., Skeers, P., Batchelor, P.E., Howells, D.W., 2013. Stem cell transplantation in traumatic spinal cord injury: a systematic review and meta-analysis of animal studies. PLoS Biol. 11 (12), e1001738. Ao, Q., Wang, A.J., Chen, G.Q., et al., 2007. Combined transplantation of neural stem cells and olfactory unsheathing cells for the repair of spinal cord injuries. Med. Hypotheses 69 (6), 1234–1237. Attar, A., Ayten, M., Ozdemir, M., Ozgencil, E., Bozkurt, M., Kaptanoglu, E., Kanpolat, Y., 2011. An attempt to treat patients who have injured spinal cords with intralesional implantation of concentrated autologous bone marrow cells. Cytotherapy 13 (1), 54–60. Bianco, P., 2014. Mesenchymal stem cells. Annu. Rev. Cell Dev. Biol. 30, 677–704. Castellanos, D.A., Tsoulfas, P., Frydel, B.R., et al., 2002. Over expression enhances survival and migration of neural stem cell transplants in the rat spinal cord. Cell Transplant. 11 (3), 297–307. Cusimano, M., Biziato, D., Brambilla, E., Donegà, M., Alfaro-Cervello, C., Snider, S., 2012. Transplanted neural stem/precursor cells instruct phagocytes and reduce secondary tissue damage in the injured spinal cord. Brain 135 (2), 447–460. Dalous, J., Larghero, J., Baud, O., 2012. Transplantation of umbilical cord-derived mesenchymal stem cells as a novel strategy to protect the central nervous system: technical aspects, preclinical studies, and clinical perspectives. Pediatr. Res. 71 (2–4), 482–490. Dao-Fang, D., San-Li, X., Ming-Ming, Z., 2009. The monolayer culturing of the neural stem cell clone and its qualification. Prog. Biochem. Biophys. 36 (1), 72–76. David, S., Aguayo, A.J., 1981. Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats. Science 214, 931–933. De Feo, D., Merlini, A., Laterza, C., Martino, G., 2012. Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection. Curr. Opin. Neurol. 25 (3), 322–333. Dlouhy, B.J., Awe, O., Rao, R.C., Kirby, P.A., Hitchon, P.W., 2014. Autograft-derived spinal cord mass following olfactory mucosal cell transplantation in a spinal cord injury patient: case report. J. Neurosurg. Spine 21 (4), 618–622. Geffner, L.F., Santacruz, P., Izurieta, M., Flor, L., Maldonado, B., Auad, A.H., Silva, F., 2008. Administration of autologous bone marrow stem cells into spinal cord injury patients via multiple routes is safe and improves their quality of life: comprehensive case studies. Cell Transplant. 17 (12), 1277–1293. Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., et al., 2003. LMO2- associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419. Iwanami, A., Kaneko, S., Nakamura, M., Kanemura, Y., Mori, H., Kobayashi, S., Yamasaki, M., Morishima, S., Ishii, H., Ando, K., Tanioka, Y., Tamaoki, N.,
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Please cite this article in press as: Muheremu, A., et al., Stem cell based therapies for spinal cord injury. Tissue Cell (2016), http://dx.doi.org/10.1016/j.tice.2016.05.008