Stroke is a leading cause of morbidity in the western world, resulting in chronic disability in many cases. Although huge efforts have been made in the search for effective treatment, so far results have been quite disappointing. Recently, cell replacement therapy has emerged as an experimental approach aiming to restore brain function, which is impaired in several neurode- generative diseases, as well as in stroke. Porcine fetal cells, neuronal progenitor cells (NPCs), embryonic stem cells (ESCs), immortalized cell lines, bone marrow stromal cells (BMSCs), and umbilical cord blood cells (UCBCs) have been introduced as potential sources for neuronal cells in experimental and clinical stroke trials.
However, limited knowledge about their biology (including long-term safety) and insufficient data regarding several issues such as the pre- ferred stroke type and its severity, specific loca- tion of the injection, and the preferred cell type restrict their potential clinical use. Therefore, further research on the molecular mechanisms of stroke, the candidate cell lines for transplan- tation, and bioengineering strategies, is needed before this technique can be implemented safely and effectively in stroke victims.
Acute Stroke
Stroke is defined as an abrupt focal loss of brain function resulting from interference with the blood supply to part of the central nervous system
(CNS). Acute stroke comprises two main types:
1) Ischemic stroke (80% of stroke cases), which is mainly caused by extracranial (large artery or car- diac) embolism or intracranial thrombosis, and 2) hemorrhagic stroke (20% of stroke cases), which is further classified to intracerebral hemor- rhage, and subarachnoid hemorrhage.
Stroke is a leading cause of mortality and mor- bidity, particularly in the middle-aged and eld- erly population, in most developed countries.1 CNS damage occurs in stroke mainly as a result of hypoxia. In the acute phase of ischemic stroke there is an ischemic gradient comprising the core, which is the central, permanently ischemic zone, and the penumbra, located at the periphery where the ischemic damage is still reversible. In the penumbra, functional alterations occur in the neurons and glial cells. Neurons are most vulnerable to ischemia because of their depend- ence on the oxidative metabolism and on glucose for energy. The pathophysiological processes in acute CNS injury such as stroke, mechanical trauma, or subarachnoid hemorrhage, are extremely complex and involve pathological per- meability of blood-brain barrier, energy failure, loss of cell ion homeostasis, acidosis, increased intracellular calcium, excitotoxicity, and free radical-mediated toxicity.1This leads to ischemic necrosis, which occurs in the regions most severely afflicted by ischemia. It can also pro- mote apoptosis, which is more likely to occur in ischemic regions afflicted less severely, evolves at a slower pace, and depends on the activation of a sequence of genes.2,3
8
Cell Replacement Therapy in Acute Stroke:
Current State
Yossi Gilgun-Sherki and Jonathan Y. Streifler
123
Although enormous amounts of efforts have been invested throughout the years aiming to provide brain tissue salvage (by using different neuroprotective agents, which were effective in animal models but so far not in humans), cur- rently the effects of acute stroke treatments are modest and chronic disability remains a major medical and economical burden on society. The only approved treatment for acute stroke today is thrombolytic therapy, which is aimed to dis- solve the occluding blood clot, thus facilitating early restoration of focal cerebral blood flow.
However, because of its short therapeutic time window (delayed administration results in increased risk of hemorrhage), this treatment is not available for most patients. Recently, there has been an increased emphasis on novel treat- ment for brain function restoration using cell replacement therapies. Indeed, many studies have shown the potential efficacy of neural transplantation in animal models of focal and global cerebral ischemia. However, very few clinical trials have been performed.
In this chapter, we discuss the potential role of cell replacement therapy in stroke, specify several cell lines as potential sources for trans- plantation, and review the factors and current limitations affecting the safety and efficacy of this method.
Cell Lines as Potential Sources for Transplantation
Various cell lines have been suggested as poten- tial sources for transplantation in stroke; the preferred line, however, has not yet been deter- mined. The molecular complexity of stroke and the involvement of different neuronal pheno- types require that the optimal cell candidate for transplantation is one that can proliferate in high numbers and differentiate into the appro- priate neural/glial cell types with maximal effi- cacy and minimal adverse events. The list of cell lines being investigated is given below.
Porcine Fetal Cells
Fetal (immature) neuronal cells can survive and integrate in the human brain, yet because of the limited availability of human tissue, some investigators have used fetal xenotransplants
from pigs, which are considered relatively safe as a donor cell source. These cells, also called lateral ganglionic eminence (LGE) cells, are pre- sumed to be undifferentiated striatal precursor cells and were first used in an animal model of Huntington’s disease in the middle of the 1990s.
It was shown that transplantation of LGE cells promoted graft integration and improved func- tional deficits.4,5 However, the extent of the capability of LGE cells to proliferate or differen- tiate has not yet been investigated and issues such as the percentage of LGE grafts differenti- ating into functional neurons versus glial cells, their plasticity, and their differentiation- dependency in the graft site have not yet been clarified.
So far, several studies have shown that trans- planted LGE neuroblasts differentiate into stri- atal neurons after intrastriatal implantation.6,7 These observations imply that LGE cells seem to be committed to follow a specific lineage pathway, and therefore cast doubts as to their use outside the striatum in the treatment of stroke.
Other concerns include graft rejection and the potential transmission of infectious diseases associated with porcine-to-human neural xeno- transplantation [e.g., the porcine endogenous retrovirus (PERV)]. However, no such evidence for clinical in vivo transmission of PERV in the blood of 24 patients with neuronal diseases who underwent transplantation with fetal porcine neurons was found.8
Neural Progenitor Cell
During the past few years, it has become clear that the mammalian adult brain contains NPCs that can proliferate, self-renew, and generate all cellular elements in the brain, including neurons. Because of the development of novel technologies, these cells can now be isolated from the subventricular zone (SVZ) and the subgranular zone of the hippocampal dentate gyrus of the developing and the adult CNS tis- sue. These cells have been shown to proliferate in response to brain damage, including focal ischemia, and therefore are a potential graft source.9–11 However, these cells cannot be transplanted in every brain region because of their limited growth support and their prolifer- ative/differentiate properties (less in the cortex than in the SVZ).12
Embryonic Stem Cells
These cells are derived from pre-embryos at the blastocyst stage and can give rise to all body tis- sues and cells. Animal models have demon- strated that ESCs, when transplanted into adult hosts, differentiate and develop into cells and tissues and thus may be applicable for treating a variety of conditions, including Parkinson’s dis- ease, multiple sclerosis, spinal injuries, stroke, and cancer. Transplanted ESCs are exposed to immune reactions similar to those acting on organ transplants; hence, immunosuppression of the recipient is generally required. It is possi- ble, however, to obtain ESCs that are genetically identical to the patient’s own cells using thera- peutic cloning techniques.13
Several studies showed that these cells are able to migrate in response to damage. Hoehn et al.14showed, using magnetic resonance imag- ing, that ESCs that were implanted into the healthy hemisphere of rat brains 2 weeks after focal cerebral ischemia (FCI), migrated along the corpus callosum to the ventricular walls, and populated en masse at the border zone of the damaged brain tissue (i.e., the hemisphere opposite to the implantation sites).
Another study showed that undifferentiated ESCs – xenotransplanted into the rat brain at the hemisphere opposite to the ischemic injury – migrated along the corpus callosum toward the damaged tissue and differentiated into neurons at the border zone of the lesion. In the homolo- gous mouse brain, the same murine ESCs did not migrate, but produced highly malignant ter- atocarcinomas at the site of implantation, inde- pendent of whether they were predifferentiated in vitro to NPCs.15
These results imply that ESCs might migrate to the damaged site. However, the production of teratocarcinoma raises concerns about the safety of ESC transplantation in patients with stroke.
Immortalized Cell Lines
ESC research raises many ethical questions and xenotransplantation limitations. Several labora- tories have therefore tracked alternative graft sources including transformed cell lines in vitro.
Two major immortalized cell lines have been used: The NTerra-2 cells and the Mandsley hip- pocampal stem cell line clone 36 (MHP36).
The NTerra-2
This cell line was derived from a human testicu- lar germ cell tumor,16 and unlike other terato- carcinoma cell lines, it has an exclusive commitment to the neuronal line when exposed to retinoic acid (RA). Several studies have shown that NTerra-2 (NT2) cells resemble NPCs. They express cell surface markers and cytoskeletal proteins unique to neural stem cells, and also yield a complement of daughter cells that retain the original phenotype.17 In addition, treatment for several weeks with RA and mitotic inhibitors produces postmitotic, neuron-like cells (NT2N), which have asymmet- rical morphology with extended axons and den- drites.17,18These cells express various neuronal characters, such as neurotransmitters,19calcium channels,20 glutamate receptors,21 and other proteins, which indicates that they have secre- tory activity and synaptogenesis.18,22 However, the major difficulties with these cells are that they cannot develop into completely full-grown neuronal phenotype in vitro, and they show full differentiation only after transplantation.23
Apparently, the survival of NT2N cells is dependent on other non-neuronal supportive cells such as glia and oligodendrocytes. Indeed, studies have showed that coculture of NT2N cells with astrocytes substantially prolongs their survival and enhances maturation and synapto- genesis in vitro, compared with NT2N culture alone.22,24
The main safety concern with undifferentiated NT2 cells is that their implantation into the rat brain leads to tumor growth and death within 10 weeks, except when they are implanted into the host striatum where grafts cease proliferating and differentiate into neuron-like cells.25,26
The Mandsley Hippocampal Stem Cell Line Clone 36
These cells are an immortalized murine stem cell line, derived from the E14 hippocampal pro- liferative zone of the tsA58 transgenic mouse.
The proliferation of this cell line is temperature- dependent, meaning that low temperatures (33˚C) result in various neuronal and glial for- mations, whereas, at high temperatures (37˚C), mature neurons and glia are produced.27
The survival of these cells after transplantation was speculated to be dependent on immunosup- pression treatment; however, Modo et al.28 showed that in transplanted post-middle cerebral artery occlusion (MCAo) rats no significant dif- ferences in cell survival were found between those treated or not treated with cyclosporine A. These results provide evidence for the low immuno- genic properties of Mandsley hippocampal stem cell line clone 36 (MHP36) cells during the initial period after implantation, which is known to be associated with an acute host immune response and ensuing graft rejection.
Bone Marrow Stromal Cells
Extraction of bone marrow (BM) gives rise to two cell populations: those that float are hematopoietic cells and those that adhere are referred to as mesenchymal stem cells (MSCs).29 These cell lines can be obtained readily from BM under local anesthesia, expanded in culture, and potentially could be delivered to injured brain tissue without the need for invasive stereotaxic operations. Using the patient’s own BMSCs should prevent host immunity and graft-versus- host disease. These cells are capable of changing their fate and differentiate into a variety of tis- sues including glia, and neurons.30–33
It was shown that when human BMSCs are exposed to neurotrophic factors (NTFs) such as epidermal growth factor (EGF) or brain-derived NTF (BDNF) in vitro or cultured with rat fetal mesencephalic or striatal cells,32they differenti- ate into NPC-like cells, and migrate along paths similar to NPCs.34
Several studies found that BMSCs from male C57 BL/6-TgN (ACTbEGFP)1Osb mice express- ing GFP that were transplanted into female C57 BL/6J mice that then underwent MCAo, became incorporated into the vasculature of the ischemic zone and expressed an endothelial cell phenotype within 3 days and at 7 and 14 days after MCAo. Some BM-derived cells also expressed the neuronal marker NeuN at 7 and 14 days after ischemia.35,36It was also shown that transplantation of nuclear fluorescence-labeled BMSCs into the ipsilateral striatum of mice sub- jected to MCAo resulted in migration into the corpus callosum and injured cortex. In addition, these cells also expressed neuronal markers, including NeuN, 4 weeks after transplantation.37
Another study showed that intravenous injec- tion of human MSC (hMSC) to rats 24 hours after MCAo revealed significant increases in numbers of enlarged and thin walled blood ves- sels and numbers of newly formed capillaries at the ischemic boundary zone (IBZ). In addition, treatment with hMSCs significantly raised endogenous rat vascular epithelial growth factor (VEGF) levels and endogenous VEGF receptor- immunoreactivity in the IBZ.38
Umbilical Cord Blood Cells
This is another potential source of multipoten- tial stem cells that can create neuronal and glial cells in response to NTFs, especially to neu- ronal growth factor and RA.39 However, our poor current knowledge regarding the biology of human UCBCs (hUCBCs) restricts their current utilization.
Cell Replacement Therapy in Acute Stroke
Experimental Data Immortalized Cell Lines
The neuron-like cells (NT2N). Several studies have examined whether grafted NT2N cells to the adult rat brain, after FCI, survive and inte- grate into the neuronal tissue. One study showed that intrastriatal transplantation of NT2N cells 1 month after MCAo, followed by cyclosporine treatment, led to significant func- tional improvement at 6 months compared with the control groups receiving either rat fetal cere- bellar grafts or growth medium alone or cyclosporine on its own.40
No studies have addressed the phenotype of NT2N grafts in the transplanted ischemic brain, nor has it been shown whether they extend processes and integrate into the host brain.
Aside from striatal injuries, the effects of NT2N cells in other ischemic infarcts remain to be determined.
MHP36 cells. Veizovic et al.41used these cells in an FCI model in rats. Transplantation was fol- lowed by immunosuppression with cyclosporine for 2–3 weeks after MCAo. They showed that
most of the MHP36 cells transplanted into the intact somatosensory cortex and striatum, remained in the intact hemisphere, whereas some (approximately 30%) migrated to the lesioned cortex and striatum. The MHP36- engrafted animals had significantly reduced infarct volumes and significantly better sensori- motor recovery over the following 18 weeks compared with sham-operated animals, whereas other parameters, such as spatial learning and memory, did not significantly improve.
Several studies have been performed at the Psychology Department in London. The first study investigated whether implantation of 200,000 MHP36 cells in the left (n = 8) or right (n = 9) brain parenchyma or their infusion into the right ventricle (n = 7) 2–3 weeks after right MCAo affected functional recovery. It was found that rats with left and right parenchymal grafts showed reduced bilateral asymmetry (i.e., improved function) but no change in spatial learning. Conversely, spatial learning improved in rats with intraventricular grafts, but a marked asymmetry persisted.42
Another recent study showed that intra- parenchymal MHP36 grafts either ipsilateral or contralateral to the lesion or intraventricular MHP36 grafts raised apolipoprotein E, a peptide which was associated with plasticity and involved in promoting functional recovery, expression levels in various brain regions 4 months after transplantation.43 These results imply that both the intact and lesioned hemi- spheres attract grafted stem cells after stroke, suggesting repair processes that utilize cells both for local repair and to augment plasticity changes in the contralateral motor pathways.
Bone Marrow Stromal Cells
BMSCs may be differentiated into neuron-like and/or glia-like cells, implying that they may have potential use in cell replacement therapy in the CNS. Eglitis et al.44(1999) demonstrated that systemically infused BMSCs into irradiated rats, subjected to FCI, migrate to the ischemic cortex and become astrocytes. Another study demon- strated that intrastriatal transplantation of BMSCs in mice 4 days after MCAo, survive, migrate, and differentiate into glial or neuronal- like cells (yet only 1% of the labeled BMSCs expressed neuronal markers45). Significant behavioral improvement at 28 days was shown
in the transplanted mice compared with con- trols, although no difference was observed in infarct volume between the groups.45
Similar results were obtained by two other studies that administered BMSCs both intra- venously and intracarotid and showed improve- ment in behavioral recovery 14 or 35 days after MCAo, respectively,46,47although only 0.02% of the cells stained for neural markers in the ischemic hemisphere.47
An additional study showed that transplanta- tion of 3 × 106BMSCs to MCAo rats improved functional recovery at day 14, compared with the controls.48A recent study demonstrated that early intervention with intravenous administra- tion of autologous BM can reduce lesion size in the rat MCAo model and improve functional outcome, as was measured by Morris water maze and treadmill stress test.49
Human Mesenchymal Stem Cells
Several studies examined the efficacy of hMSCs in the treatment of experimental stroke. One study reported a significant functional recovery in rats treated with hMSCs at 14 days compared with control rats in a transient MCAo model.
Furthermore, few (1%–5%) hMSCs expressed proteins phenotypic of brain parenchymal cells.50
An additional study showed that purified hMSCs that were grafted into the cortex sur- rounding the area of infarction, 1 week after cor- tical brain ischemia in rats, improved functional performance in several behavior tests. In addi- tion, histologic analyses revealed that trans- planted hMSCs expressed markers for astrocytes (GFAP), oligodendrocytes (GalC), and neurons (beta III, NF160, NF200, hNSE, and hNF70).
Therefore, it is unlikely that the functional recov- ery observed in the ischemic rats, which received hMSC grafts, was mediated by the integration of new “neuronal” cells into the circuitry of the host brain. Rather, these results imply that the func- tional improvement might have been mediated by proteins secreted by the transplanted hMSCs, which could have up-regulated host brain plas- ticity in response to experimental stroke.51
Another recent study showed that intra- venous injection of hMSCs to rats that under- went MCAo improved their motor behavior, reduced apoptosis, and promoted endogenous cell proliferation.52
According to the preliminary results regard- ing BMSC transplantation in stroke, it seems likely that these cells might become good candi- dates for the treatment of stroke. However, because of our limited knowledge on neuronal differentiation, there are many unknown factors such as the capability of these cells to differenti- ate into mature neurons that are capable of reconstructing the natural neuronal circuitry, and other concerns such as the adequate cell number for transplantation (see Table 8.1) and brain targeting.46
Umbilical Cord Blood Cells
UCBCS are the source of hematopoietic stem cells and other stem and progenitor cells. Chen et al.46(2001) reported that intravenous infusion of 3 × 106hUCBCs to rats 24 hours after MCAo, improved behavioral recovery 14 days after implantation compared with control animals subjected to FCI alone or FCI with saline injec- tion. An estimated 2% of these cells expressed neuronal markers, whereas an unknown per- centage survived and migrated to the ischemic hemisphere, and also to various other organs.46
Another recent study assessed which route of hUCBC administration, intravenously into the femoral vein or directly into the striatum, might produce the greatest behavioral recovery in rats
with permanent MCAo. It was found that the spontaneous activity decreased significantly when hUCBCs were transplanted 24 hours after stroke compared with nontransplanted, stroked animals. In addition, in the step test, a signifi- cant improvement was found only after femoral delivery of the hUCBCs. This was in contrast to the behavioral recovery that was similar in both striatal and femoral hUCBCs delivery.53 These results suggest that the intravenous route might be more effective than the striatal route in pro- ducing long-term functional benefits to the ani- mals after a stroke.
This source of cells is interesting because of ready availability and it does not raise ethical issues. Although functional improvement has been demonstrated in animal stroke models, much work lies ahead in order to determine unequivocally whether UCBSCs will serve as a potential source for cell transplantation.
Clinical Trials
To date, only one clinical study has been reported, using cultured human neuronal cells (NT2D1, RA treated) in patients with stroke.
This was an open, phase I clinical trial that assessed the safety and feasibility of human neu- ronal cellular transplantation in patients with basal ganglia stroke and fixed motor deficits. It included 12 patients (aged 44–75 years) with an infarct 6 months to 6 years old (and stable for at least 2 months).54Four of the 12 subjects were implanted with 2 million cells, divided into three implants, whereas the others received 2 or 6 mil- lion cells (at one or three-pass injections), at random. Serial evaluations (12–18 months) showed no adverse cell-related serologic or imaging-defined effects.54The European Stroke Scale score was significantly improved in six patients (3 to 10 points), with a mean improve- ment of 2.9 points in all patients. Motor per- formance accounted for most of the change, being better in the 6-million cell group. Both the Barthel index and the SF-36 disabilities scales were not consistent as they showed improve- ment only in the 6-million cell group. In addi- tion, positron emission tomography (PET) scanning at 6 months54and 12 months55showed greater than 15% relative uptake of 18F-fluo- rodeoxyglucose (FDG) at the transplant site in six patients. These findings, including the FDG- PET scan, are encouraging; however, because of
Table 8.1. Possible factors that influence the efficacy and safety of transplantation in stroke
• Isolation and expansion techniques
• Number and volume of transplanted cells
• Cell medium (e.g., neurotrophic factors)
• Cell type
• Cell survival (e.g., neurotrophic factors)
• Cell integration (e.g., neurotrophic factors)
• Surgical technique
• Postoperative care
• Place of transplantation and area-specific brain targeting
• Blood supply to the injection site
• Stroke type (ischemic versus hemorrhagic)
• Time of transplantation
• Severity of the damage
• Age and gender of the patient
• Graft rejection and cotherapies such as immunosuppressants
• Appearance of tumors (teratogenicity)
• Co-morbidities and especially other neurological diseases
the small number of patients included in these studies, definitive conclusions cannot be drawn regarding the clinical efficacy.
Factors that Might Affect Efficacy and Safety
Technical Considerations
There are many factors that might influence the efficacy and safety of cell transplantation in stroke (Table 8.1). Among them are the numbers of cells transplanted, which is still relatively unknown. This issue is also important for plan- ning delivery methods given that the different volumes that may be needed for adequate cell numbers may require different methods.
Another important factor is the type of cell to be transplanted. This is of cardinal importance because of the effectiveness and safety proper- ties of each cell type. For example, it is well known that several cell lines (e.g., ESCs) form fatal teratomas at the transplantation site.
Tumor formation would be a serious disadvan- tage to transplantation in humans.
An additional major issue is the site of trans- plantation as it involves other factors, such as blood supply to the injection site and the stroke type (ischemic versus hemorrhagic). Cells might be implanted intraventricularly or into brain parenchyma. It is reasonable to suggest that the penumbra is the preferred area for cell implan- tation, because of a reduced content of excito- toxicity and toxic substances such as proinflammatory cytokines and reactive oxygen species and better blood supply through collat- eral circulation, but this issue requires further examination. In addition, several cell lines can- not be transplanted in every brain region, because of limited growth support (e.g., NPCs are known to be less proliferative in the cortex than in the SVZ).
The timing of the transplantation is also an important factor that might affect the success of transplantation. The optimal timing for a trans- plant is unknown, yet stroke type, chronicity, and also blood supply (inadequate blood flow would not support graft viability) must be taken into account. The severity of the damage and natural course of recovery from the stroke must also be considered. Many neurologists prefer to
transplant at least 1 month after the stroke and preferably when deficits reach a plateau.
However, during that delay, the formation of scar tissue may occur, which might adversely affect the implanted grafts. The age of the patient also influences the efficacy of the trans- plantation, and it is reasonable to presume that younger patients would respond differently than older ones. In addition, the gender and hor- monal status of the patient might also have impact on cell survival. Finally, the use of immunosuppressants should also be taken into consideration because they might complicate the interpretation of the therapeutic effect.56
Preimplantation Manipulations
These procedures are speculated to be very cru- cial in the survival of the cells posttransplanta- tion, and might produce better-characterized neuronal donor populations. In addition, they can help to identify the donor glial-neuronal interactions,57 and can assist in understanding molecular pathways in stroke, such as apoptosis in pre- and posttransplantation.58Indeed, apop- tosis inhibitors were shown to improve the via- bility of the grafts when they were administered before or with the implanted cells.59,60
One of the most important factors that might contribute to cell survival and integration after transplantation is NTFs, which are naturally secreted peptides from neurons and neuron- supporting cells that act as growth factors for the development, maintenance, repair, and sur- vival of specific neuronal populations61,62 (see also Table 8.1). These factors stimulate axonal growth and have a key role in the construction of the normal synaptic network during develop- ment.63,64 However, in aging and pathological states, such as head trauma, chronic (e.g., neu- rodegenerative diseases) or acute (e.g., stroke) diseases, these factors might be deficient, and therefore influence neuronal survival leading to apoptosis.61,65–67
Several recent studies support these concepts:
A composite graft of fresh BM along with BDNF, transplanted into the IBZ of rat brain, facilitated BM cells to survive and differentiate and improved functional recovery.68 BDNF and nerve growth factor were also shown to decrease significantly apoptotic cells in the IBZ of the ischemic hemisphere of rats treated with hMSCs.50
Another study demonstrated that basic fibroblast growth factor-secreting cell line that was implanted into the right striatum of rats 6 days before MCAo showed a 30% reduction in the infarct volume compared with the controls with encapsulated naive baby hamster kidney cell grafting or those without implantation at all.69,70
In a different study, four clones of fetal rat kidney cells producing GDNF at physiological concentrations were transplanted into the ischemic core or penumbra of rats that had undergone MCAo. Three of these four clones showed a reduced volume of infarction and improved behavioral abnormalities as com- pared with controls.71
These results suggest that supplementation of NTFs, either pre-, parallel, or post-transplanta- tion, supports the surviving cells or enhances the local environment to improve function, and therefore might be beneficial in the treatment of stroke.
Conclusions and Future Strategies
A variety of cell types have been used to try to restore brain function after stroke, mainly in rodent models. Some of the findings presented herein imply that cell replacement therapy might indeed ultimately become a novel restora- tive therapy for stroke. However, because of our limited knowledge on the molecular basis of stroke and cell transplantation, there are many open questions regarding the efficacy (e.g., graft integration, survival, and functionality; see Table 8.1) and safety (e.g., tumor formation) of each cell source. At present, it is also still debat- able whether the cells themselves integrate and function as active neurons and glia (i.e., form new neural pathways and connections) or they mainly act through neurohumoral mechanisms (i.e., stimulation of intrinsic host recovery mechanisms). Trophic factors are probably needed and may be even more important than the cell type or vehicles used. It is likely that multiple mechanisms are involved. Therefore, long-term investigations into safety and effi- cacy, together with comparative studies between cell types, and different trophic factors, are still warranted. They will enhance our understand- ing of cell replacement therapy in stroke, and facilitate its application in clinical practice.
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