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Cellular replacement and regenerative medicine therapies in ischemic stroke Worldwide, tissue engineering and cellular replacement therapies are at the forefront of the regenerative medicine agenda, and researchers are addressing key diseases, including diabetes, stroke and neurological disorders. It is becoming evident that neurological cell therapy is a necessarily complex endeavor. The brain as a cellular environment is complex, with diverse cell populations, including specialized neurons (e.g., dopaminergic, motor and glutamatergic neurons), each with specific functions. The population also contains glial cells (astrocytes and oligodendrocytes) that offer the supportive network for neuronal function. Neurological disorders have wide and varied pathologies; they can affect predominantly one cell type or a multitude of cell types, which is the case for ischemic stroke. Both neuronal and glial cells are affected by stroke and, depending on the region of the brain affected, different specialized cells are influenced. This review will address currently available therapies and focus on the application and potential of cell replacement, including stem cells and immortalized cell line-derived neurons as regenerative therapies for ischemic stroke, addressing current advances and challenges ahead. KEYWORDS: adult stem cell n bone marrow-derived cell n cellular therapy n immortalized cell line n ischemic stroke n neural stem cell n NT2N

Neurological disorders are a degree of complexity greater than other afflictions suffered by humans due to the multifunctional nature of the brain and the heterogeneous populations of cells that maintain this. Neurological disorders at present have limited treatments. The available treatments are either drug or surgical techniques, which work in a palliative or damage limitation manner, but they do not slow or repair the damage. This presents an attractive focus for regenerative medicine and the use of transplanted cells. Treatments that are being developed in laboratories may function in a neuroprotective or tissue-replacement manner. In order for stem cell therapies to be considered as a replacement for conventional treatments, they need to fulfill certain criteria. They need to be safe, long-lasting and present substantial improvements over currently available therapies, as well as being commercially competitive. Knowledge in this area is steadily progressing, and with our advancing ability to mimic human disorders in animal models [1] , we are now betterequipped to understand the nature of these diseases and their pathologies. That said, there is still a large amount of research to be done before stem cell technologies can be fully implemented into clinics. Therapies in the development phase will have to be specifically designed with a disorder in mind. Different disorders and diseases require different cell types, whether this is an individual cell type

or a heterogeneous cell population. The donor cell type must be decided. This could either be auto logous or allogeneic; both autologous and allogeneic cells have their own advantages and disadvantages associated with them. In some cases, the use of allogeneic, in particular human embryonic stem (hES) cells, is fraught with ethical constraints and controversy. As these cells are harvested from surplus in vitro fertilized embryos, there are very strong concerns about the moralistic value of destroying early human embryos. Autologous cells bypass many of these ethical issues as they are harvested from the recipient patient. Current treatments for ischemic stroke are limited with regard to their function and availability. Having only limited administration windows, the majority of sufferers do not benefit from the available therapies, due to late diagnosis and treatment. Within regenerative medicine, there is the potential that stem cell replacement could be used early in a neuroprotective capacity or as a cell-replacement strategy following the occurrence of stroke. It is hoped that eventually we will be able to move away from palliative and damage limitation treatments and move more towards long-term organ function regeneration.

10.2217/RME.12.2 © 2012 Future Medicine Ltd

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John W Thwaites1,2, Vikash Reebye2, Paul Mintz2, Natasa Levicar2 & Nagy Habib*2 Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK 2 Division of Surgery & Cancer, HPB Unit, Hammersmith Hospital, Imperial College, London, UK *Author for correspondence: Tel.: +44 20 3313 8574 Fax: +44 20 3313 3212 [email protected] 1

Ischemic stroke Stroke is the third highest cause of mortality globally, and is a leading cause of long- and shortterm disability [2] . Of those that survive the first

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year post-stroke, up to 50% will be left with permanent disabilities [3] . The financial burden of this disease is huge, including both the immediate and continued healthcare for stroke patients. The estimated cost of this in the UK alone is thought to be approximately £2.8 billion annually [4] . As stroke affects people later in life and the average life expectancy is on the increase, this problem is predicted to worsen. For this reason, long-term stroke therapy is an area that needs to be addressed, and it is hoped that this can be done through the use of therapeutic stem cells. Stroke is associated with many risk factors, including medical, social and genetic [3] . There are two main types of stroke: ischemic and hemorrhagic. Both types of stroke lead to disruption in blood flow to an area of the brain, resulting in cell death. Ischemic stroke accounts for approximately 75–80% of all strokes [5] and is caused by severe narrowing of the arteries by over 50% [3] . The brain is extremely susceptible to damage due to its highly vascularized structure and its large requirement for oxygen to fuel its metabolic function; this is reflected by the fact that approximately 20% of the oxygen found in the circulatory system is utilized by the brain [3] . A major cause of ischemic stroke is atherosclerosis, which is the build up of plaque in the lumen of the blood vessels, leading to stenosis. Sequentially to this is the build up of a thrombus at the site of artherosclerosis or remotely where it migrates to the site of the infarct, resulting in an embolism [3] . The drug and mechanical treatments currently available or under development for stroke patients have a major limitation in that they all have a restricted administration window in order to be effective. Currently, there is only one approved drug treatment commonly used at the onset of stroke. This is intravenous recombinant tissue plasminogen activator [2] , approved in 1996 by the US FDA [6–8] . Recombinant tissue plasminogen activator acts as a thrombolytic agent and has a functional treatment window of 6 h from the onset of stroke, although it should ideally be administered within 90 min of the onset. Due to this restricted administration window, only 3–4% of stroke sufferers benefit from the available treatment [4] . If stroke victims have a significantly delayed intervention, the damaged tissue at the core of the stroke region ultimately begins to necrose, thus causing secondary damage to the surrounding penumbra. Other drug therapies are under trial, but with limited success. Many compounds focus on thrombolytics and the breakdown of the insulting thrombus, but these are affective only within 388

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a specific timeframe from onset. Additional drugs that have been investigated include antiplatelets and anticoagulants, but these also have limited benefits on revascularization of the affected area. These classes of drugs work mainly as a prophylactic adjuvant for those who are at high risk of developing a stroke, or as a preventative measure for recurrence in patients who have experienced a stroke [9–12] . In addition to pharmacological intervention, mechanical means of stroke treatment are also being investigated with some success. This form of intervention requires the use of a mechanical device that physically targets the thrombus for its removal from the vessel. FDA-approved devices include: the Mechanical Embolus Removal of Cerebral Ischemia (MERCI® ; Concentric Medical, CA, USA) and a mechanical thrombectomy device, the Penumbra System® (Penumbra, CA, USA) [8,13] . Both of these devices have an 8‑h treatment window in order for their use to have a beneficial effect on revascularization, resulting in a positive neurological outcome [14–16] .

Cellular therapies for ischemic stroke With the limitations associated of current therapies the advent of cell transplantation as a means of long-term regeneration therapy is an attractive adjuvant for stroke patients. Stem cell therapies could act in a trophic, neuroprotective capacity, reducing the damage site and aiding in endo genous neurogenesis. Cells could also be administered at a later stage to replace nonviable tissues and restore function. Many aspects need to be taken into account when considering cellular replacement as a treatment. Individual areas of the brain contain unique specialized cell populations that will dictate the specific cellular requirements for transplantation at that site. Infarct size will dictate the volume of cells required; this will inevitably mean that a standardized dose regime is unlikely to be implemented. These factors result in treatment plans that are tailored around the needs of the individual patient [17] . Timing of the treatment will depend on the microenvironment of the tissues surrounding the affected area, which will be constantly changing following an ischemic event, with a deregulated surge in proinflammatory mediators, excitotoxic neurotransmitters and free radicals [18,19] . The presence of these factors will therefore have to be considered when transplanting the therapeutic stem cells. Finally, and perhaps the most important consideration, would be the choice of site for transplantation, with it being desirable to opt for the most efficient delivery in future science group

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the shortest time possible. The choices range from a systemic intravenous delivery to a more targeted approach via the fluid-filled sites of the infarct, directly into the core of the penumbra or collaterally to an unaffected side of the brain to allow cells to migrate to the damaged sites [20,21] . Since these questions have not yet been fully addressed, any investigation into this area will undoubtedly contribute to pushing the boundaries of regenerative therapy closer to reality. The pace of stem cell research in the field of cellular reprogramming for regenerative medicine has accelerated at a breathtaking rate. Investigators worldwide now have the ability to reprogram both adult somatic and stem cells to create tailor-made cell types, including those of the neuronal lineage. These cells include: hES cells [22–25] , the immortalized cell line NTera2 (NT2) [26–29] , adult cells, including fibroblasts [30] , bone marrow-derived cells [31] and cells from adipose tissue [32] . Other stem cells, such as hematopoietic stem cells (HSCs), have been shown to possess a neuroprotective capacity in vivo [21,33,34] . Since cell reprogramming has now clearly evolved from a niche expertise to a more common technique used by researchers in a multitude of fields, numerous constraints must now be met in order to standardize the process so that this form of technology becomes successfully incorporated as a therapeutic alternative. Cells that are used must be easily expandable and, once differentiated to their specific lineages, must be able to maintain stable progenitors without the danger of becoming tumorigenic.

In vitro reprogramming of cells into neurons hES cells are an attractive source of cells for in vitro differentiation due to their capacity to expanded indefinitely both in their primitive stage as well as their differentiated stage. Reubinoff et al. demonstrated that it was possible to generate an enriched population of neuronal progenitors from the HES-1 cell line [23] ; from these progenitors, they were able to promote undifferentiated proliferation of the neuronal precursors. Similarly, Chambers et al. was able to differentiate 80% of cultured hES cells into functional neurons by using two inhibitors of Smad signaling (intracellular proteins that transduce extracellular signaling), Noggin and SB431542 [22] . Through a process of patterning induced by the addition of selective growth factors, including BDNF, ascorbic acid, GDNF, TGF-b1, SHH and FGF8, Chambers et al. were able to derive mature motor and dopamine-producing neurons [22,25,35] . future science group

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Despite this achievement, differentiated hES cells have still not met the necessary requirements for implementation into a clinical protocol for the treatment of ischemic stroke. However, trials in other CNS-associated diseases and trauma, including spinal cord injuries, macular dystrophy and spinal muscular atrophy, have been approved [36] . There is a caveat associated with the use of embryonic-derived stem cells in therapy, as they possess a strong potential to form teratomas [37] , and their culture typically requires the use of xeno-recombinant-derived products, which carries the risk of trans-species viral transfer. This markedly restricts their clinical usefulness. With this in mind, researchers are exploring alternative sources and methods of generating therapeutically useful cells. These include immortalized cell lines [26–29] , neural stem cells (NSCs), bone marrow-derived cells – mesenchymal stem cells (MSCs) and HSCs [38,39] – and adipose tissue-derived stromal cells [32] . The functional potential of cells that have already been stably differentiated towards the neuronal lineage are still being characterized in the hope that they will shed light on the long-term behavior of such induced cells. A good example of this can be found with the NT2 cell line, which was previously established from an adult human testicular germ cell tumor [26] . NT2 has been shown to be exclusively committed to differentiate towards the neuronal lineage upon exposure to retinoic acid (RA), where the induced cells (NT2N) display functional neuronal characteristics [27–29] . Once differentiated, NT2N cells show evidence of expressing neurotransmitters [40] , glutamate receptors [41] , calcium channels [42] , mature cytoskeleton and neurofilament proteins [43] . This indicates that viable, functional neuron-like cells can be generated from cells that pertain to a separate lineage. An example that demonstrates the plasticity of adult stem cells can be seen in studies using bone marrow-derived cells. Woodbury et al. demonstrated that the bone marrow stem cell sublineage, MSCs, can be induced to differentiate into neurons in an in vitro setting [44] . The group cultured MSCs in the presence of differentiation factors that included b-mercaptoethanol and dimethylsulfoxide. They observed that these cultured MSCs began to express the neuronal markers, such as neuron-specific endonuclease, NeuN and neurofilament-M. Deng et al. also showed that it was possible to differentiate at least 25% of a total population of cultured MSCs into neuronal progenitors, simply by increasing its intracellular levels of cAMP [45] . Although www.futuremedicine.com

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Deng et al. observed a similar change in their differentiated adult stem cells when compared with the Woodbury et al. group, their differentiated cells did not express mature markers of neuronal cells such as neurofilament-M or GFAP. It is clear that adult stem cells have the potential to be redirected towards a neuronal lineage; however, other factors are required to completely commit these cells to the relevant differentiation pathway. Hermann et al. showed that MSCs were able to differentiate into neuronal-like progenitors when cultured with FGF2 and EGF [46] . Further differentiation using BDNF and RA allowed these neuronal-like MSCs to produce mature neuronal cell types expressing all the necessary markers, including tyrosine hydroxylase, GFAP and b-III-tubulin. Adult human fibroblasts have also been successfully reprogrammed into neurons through forced expression of specific genes. Pfisterer et al. demonstrated that the transient upregulation of four target genes was sufficient to reprogram human adult fibroblasts into neurons at a conversion efficiency of 16%, of which 95% expressed the mature neuronal protein MAP2 and the synapse formation marker synaptophysin (a peptide involved in synaptic transmission) [30,47] . These studies demonstrate that stem cell populations other than hES cells may be used to produce cells of neuronal lineage. It is hoped that our understanding of adult stem cells has advanced sufficiently to allow researchers to move away from controversial cell types and move towards the use of potentially patient-specific cell sources.

In vivo potential of stem cells in stroke NT2N cells Following the successful establishment of a differentiated neuronal cell line (NT2N) by the Andrews and Damjanov laboratory [48] , it was necessary to address the next challenge in successfully transplanting these cells into a live animal model with a functional output. It was found that NT2 cells implanted into nude mouse models induced metastatic tumor formation with the exception of when they were implanted into the striatum (area of high RA levels) [49,50] . NT2N cells (NT2 cells exposed to RA) showed no evidence of tumor or neoplastic growth in mouse models of stroke over a period of at least 14 months [48] ; this result could be attributed to the fact that NT2 cells may lose their proliferative capacity once exposed to RA and therefore continue their expansion towards the NT2N variant. The observations from this study made it clear that the microenvironment of 390

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the tissues surrounding the site of transplantation would have to be fully characterized to ensure a successful and, in the long term, risk-free grafting [51] . Since allogeneic cell transplantation would normally require adjuvant immunosuppression of the recipient to prevent donor–host rejection, it was surprising to observe that this was not an issue following transplantation of NT2N cells into human subjects. It was established that protection against the host’s immune system was conferred onto NT2N cells by its expression of the cell surface peptides CD59 and Kappa [52,53] . The ability of NT2N cells to survive transplantation in vivo can also be attributed to enhanced expression of the neuroprotective factor, GDNF. These features combined allow NT2N cells to integrate into the host tissue, demonstrating its regenerative capability [28,49,51] . Bone marrow-derived cells A number of preclinical trials have taken place to study the impact of using autologously derived adult stem cells within a cerebral ischemic setting [54] . Bone marrow-derived cells are known to be developmentally versatile and also have the advantage of being sourced autologously from the recipient patient. The Chen group showed that rats subjected to a middle cerebral artery occlusion (MCAO) for 2 h gained significant recovery when injected intravenously with rat-derived MSCs at 24 h and 7 days postMCAO when compared with control animals [55] . They showed that a significant number of transplanted MSCs remained viable within the host and had migrated to the ipsilateral ischemic hemisphere. They demonstrated that infusion of cells at both time points resulted in significant behavioral improvements (neurological severity scores, adhesive removal and rotarod tests) in the MSC-treated groups compared with untreated controls [55] . A similar observation was made by Shen et al. [56] . Rats subjected to MCAO were administered with rat-derived bone marrow aspirate that was rich in MSCs 1 month following arterial occlusion. The treated animals showed significant behavioral recovery when compared with the control group. These rats were then sacrificed 4 months following the procedure, where the transplanted cells were found to be localized to the sites of injury, as well as showing an upregulation in expression of the astrocyte marker GFAP, the mature neuronal marker MAP2 and synaptophysin. Additionally, there was a significant reduction in scar tissue around the ischemic region, with a higher frequency of proliferating resident cells. future science group


Bone marrow-derived HSCs isolated directly from bone marrow biopsies or mobilized using G-CSF [57,58] have been investigated for their potential use in neuronal regeneration. HSCs demonstrate a ‘homing in’ ability towards sites of tissue injury while secreting compounds such as VEGF, FGF2 and IGF1, which can aid in revascularization and protection of damaged tissue [21,59] . Taguchi et al. demonstrated this by using mice models with surgically occluded middle cerebral arteries (MCAs) [21] . Injection of HSCs into these injured mice showed enhanced angiogenesis and neurogenesis at the sites of cerebral infarction. The HSC-treated group also demonstrated less behavioral abnormality when compared with the untreated control group [21] . Schwarting et al. also investigated the anti-inflammatory and trophic value of injecting HSCs into mice with transient MCAO [33] . The injured mice were treated with purified HSCs 24 h after the occlusion and were sacrificed 24 h thereafter for post-mortem investigation. The HSCs had migrated to the peri-infarct region, where they showed expression of microglial proteins but no expression of neuronal markers. Tissue injury analysis showed that the treated mice had significantly smaller infarct volumes and lower levels of apoptotic cell death in the peri-infarct area when compared with the untreated control group. When analyzing the immune response of the treated mice, it was shown that the HSCs appeared to suppress gene transcription of the proinflammatory genes at the spleen, thus lowering the host immune response [33] . Although there have been observed benefits of administering HSCs after an ischemic attack, it is still unknown whether these cells can migrate to other organs of the body, thereby causing unwanted side effects through heterotypic cell fusion. Shyu et al. addressed whether G-CSF-induced mobilization of HSCs would be beneficial in an ischemic animal model [34] . Rats were subjected to occlusion of the MCA and carotid artery to induce cerebral infarction for a period of 90 min before reperfusion was re-established. The experimental group was given G-CSF over a period of 5 days. Treated rats showed enhanced revascularization and neural repair at the site of the infarct when compared with the untreated control group. There was also a reduction in the infarct volume and enhanced recovery of neurological function in the treated group. This work also demonstrated that chemokines originating from the site of injury allowed mobilization of the circulating HSCs across the blood–brain barrier. future science group

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Stromal adipose cells Adipose tissue-derived adult cells have been shown to have a capacity for differentiation towards a neuronal lineage. Kang et al. used human adipose stromal cells isolated from liposuction tissue and exposed these cells to a defined differentiation protocol [32] . The stromal cells showed an upregulation in the expression of mature neuronal markers MAP2 and GFAP. Rats subjected to MCAO were then transplanted with these differentiated stromal cells 24 h postocclusion. A significant improvement in motor function was observed in the treated group compared with untreated controls. Post-mortem studies of mice at 14 and 30 days following treatment showed that the transplanted cells were located in the brain, particularly at sites of ischemic damage [32] . Later work by Du et al. showed that transplantation of nonmanipulated adipose tissue in MCAO models led to enhanced expression levels of IL-10 (an anti-inflammatory and survival factor) around the damaged site, suggesting that adipose stromal cells are able to confer a neuroprotective role [60] . Neural stem cells Human NSCs have also been proposed for use in neuronal regeneration. These cells are derived from the embryonic or fetal CNS. Kelly et al. used human neurospheres cultured from isolated NSCs and implanted these cells directly into the cortex of rats subjected to MCAO [61] . The animals were sacrificed for analysis 4 weeks following intervention. Immunohistological staining showed that the implanted NSCs only survived in nonischemic tissues. Cells transplanted into the damaged sites did not survive; however, there was evidence of cells migrating from the nonischemic area towards the ischemic zones. Cells that had successfully migrated within the damaged zone showed evidence of induced differentiation, as they expressed the relevant markers, including b-III-tubulin and GFAP [61] . Similar work by Darsalia et al. showed robust survival of implanted neurons in MACO-induced rats [62] . Migrating NSCs showed upregulation in mature neuronal markers, such as HuD, calbindin and parvalbumin, while the nonmigrating NSCs remained in a more basal state, with very low expression levels of the neural marker nestin and immature neuronal lineage markers b-III-tubulin and GFAP [62] . Although this work demonstrates the potential use of this cell type, its routine use in a clinical setting is questionable due to being sourced from embryo or fetal tissue. This raises both ethical concerns, as well as logistical issues associated with availability and quality control. www.futuremedicine.com

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Clinical studies NT2N NT2N cells have been investigated in Phase I safety trials. A 2002 study using 12 patients that had suffered basal ganglia infarcts between 6 months and 6 years previously were immunosuppressed and injected with a defined number of NT2N cells [63] . The engraftments did not show any adverse effects, and the one patient that died during the study from an unrelated cause demonstrated that the transplanted cells survived for up to 27 months following implantation [63] . A randomized, Phase II, open-label trial was carried out in 2005 to study both the safety and efficacy of NT2N treatment in 14 patients with both ischemic and hemorrhagic stroke [64] . The inclusion criterion was that all patients showed stable motor defects between 1 and 6 years following their infarction. The patients were transplanted with a defined number of NT2N cells, and no adverse reactions were observed. Assessment with various stroke-scoring systems, including the European Stroke, Health Stroke and Stroke Impact Scales, were performed preimplantation and at 6 months postimplantation. Analysis of the data showed a trend towards improvement in the patient groups who had received the NT2N treatment. This, however, was not significant, as it was concluded that the study size was too small and the inclusion of hemorrhagic patients may have compromised the accurate analysis of the study [19,65] . Bone marrow-derived cells The inclusion of bone marrow-derived cells in clinical trials was first reported in 2005. Bang et al. conducted a Phase I trial investigating the safety and feasibility of autologous, in vitro-expanded MSCs in 30 patients with cerebral infarcts within the MCA [66] . Patients were randomly assigned to either the treated group or control group. The patients were injected intravenously with a defined number of autologous cells, and neurological examinations were performed in both groups of patients using the Barthel Index and Rankin Score 1 year postimplantation. It was found that the treated group steadily improved in both the index and score during the follow-up period when compared with the control group. There were no adverse reactions reported following cell infusion, and so it was concluded that this procedure was sufficiently safe to continue. A similar observation was also reported from a long-term Phase I study involving 52 patients [67] . The inclusion criteria were for patients that had suffered a severe infarct of the MCA and were followed up for 5 years post-cell transplantation. 392

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The results showed no significant side effects in the patient group that had received the cell infusion, and comorbidity was equal in both control and treated patients. Assessment with the modified Rankin Score showed clinical improvements in the treated patient group when compared with the control group. This improvement was associated with serum levels of SDF-1 and involvement of the subventricular region of the lateral ventricle. This study therefore confirmed the safety and feasibility of using MSCs for treatment. Suárez-Monteagudo et al. performed safety trials, injecting cells obtained from autologous bone marrow samples; these were stereotactically implanted along tracts of the perilesional region in five patients [68] . The study showed no adverse effects from the treatment, with some neurological improvements observed 1 year after the procedure. Although the results showed trends towards improvement, no conclusions could be drawn due to the small sample size. A recent study by Savitz et al. involved ten patients aged between 18 and 80 years who had suffered acute MCA ischemic stroke who were injected with autologous mononuclear cells [69] . Patients were treated successfully with no adverse events. There were no study-related adverse effects, but one patient died 40 days post-stroke from nonprocedure-related complications. Analysis of the surviving recipients showed all patients improved, with seven of the patients achieving greater that 90 on the Barthel Index. This study concluded that the use of mononuclear cells is safe and feasible in therapy for acute stroke. A number of other Phase I and II clinical studies are underway to assess the therapeutic benefits of using bone marrow-derived HSCs. These trials will again determine the safety and efficacy of the procedure, but will also establish the appropriate administration times that can be used to achieve the desired clinical outcome. Cells will be administered to patients 7 days (ten patients), 5–9 days (20 patients) and 6–60 months (30 patients) post-stroke [4,70] . Neural stem cells The first regulated Phase I clinical trial for the treatment of stroke, PISCES, using NSCs received approval from the Gene Therapy Advisory Committee (GTAC, UK) and is now under way [101] . This study will establish the safety and feasibility of using the ReN001 immortalized NSC line, derived from human fetal brain tissues, for stroke treatment. Twelve patients not showing clinical improvement 6–24 months following a stroke event have been recruited for this future science group


trial. The first five patients out of this group have already been injected with a low dose of ReN001 and will undergo frequent reviews over a period of 2 years. During this time, the other seven patients will follow the same therapy but with increasing numbers of cells [71] . The results of these studies will be eagerly awaited, as it will pave the way for other groups to focus on developing regenerative stem cells across a wider range of therapies.

Conclusion Research into therapeutics for stroke has progressed immensely over just half a decade. This undoubtedly is indicative that future studies will be exciting and will hold promising prospects for not only stroke therapy, but also for other neurological disorders. While we confirm that these cells are safe and feasible for long-term patient care and we establish a standardized route towards incorporating this into a robust treatment plan, scientists are racing to fully unravel the true potential of stem cells. We are only just beginning to appreciate how this will revolutionize the face of ‘tailor-made’ medicine. Future perspective Regenerative medicine is a fast growing area of research. To date, researchers are able to differentiate a number of cell types into neurons, this has gone from a niche expertise to a widely used laboratory technique. Work has now moved to investigating these differentiated cell types in vivo and looking at their efficacy and safety. Clinical application of this knowledge has been slow to date, although we are now seeing the research produce clinical deliverables. A

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trial in the UK has started using immortalized NSCs (PISCES) and there are a number of trials ongoing using bone marrow-derived adult stem cells, looking at safety and efficacy. With both research and clinical trials progressing, the next decade is going to be an exciting time for regenerative medicine in healthcare. This, though, is only going to happen if investment in this area continues, allowing continued research and the application of these new therapies. Collaboration is also going to key to their development, both between academic institutions but also between industry and academia. The development of these therapies is going to be dependent on multicenter and international collaboration. Alhough it is a very exciting area of growth, it is also important to keep in mind that we do not yet fully understand a lot of the diseases we aim to treat using regenerative medicine. Though our knowledge is increasing, caution must be taken not to pursue this opportunity in a carefree manner. Research needs to continue in both the development of these therapies but also in the basic science that allows us to understand and to properly treat. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Background Current treatment regimes for patients that have suffered ischemic stroke only function in a palliative manner. It is hoped that through the use of stem cells and cell-replacement therapies, treatment can move towards a cure. In vitro reprogramming of cells into neurons Human embryonic stem cells provide an attractive source of cells, but due to ethical constraints and the chance of uncontrolled differentiation and tumor development, researchers are investigating other cell sources for replacement therapy. It has been shown that it is possible to selectively reprogram a variety of cell types, including adult stem cells and immortalized somatic cell lines, into neuronal cell types. In vivo potential of stem cells in stroke In vivo studies have indicated the therapeutic potential of a variety of cell types, including bone marrow-derived cells (mesenchymal stem cells and hematopoietic stem cells), somatic adipose cells and neural stem cells. Clinical studies Clinical trails are underway to study the therapeutic potential of a variety of cell types, including mesenchymal stem cells and hematopoietic stem cells. The first regulated Phase I clinical trial for the treatment of stroke is underway, using immortalized neural stem cells, and the results are eagerly awaited.

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