Stem+Cell+Therapy+in+Stroke

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=**1. Introduction to Stem Cell Therapy for Stroke **= A stroke leads to widespread loss of neurons and glial, causing neurodegeneration, paralysis or death. Current stroke treatments such as thrombolytic therapy, have a small time frame in which to work after the stroke occurrence, and only a small percentage of patients benefit from this treatment, rendering them fairly ineffective. Neuro-regeneration which occurs up till weeks after the stroke, allowing a larger time window for treatment, can be __done__ through transplantation of exogenous __stem cells__ or proliferation and differentiation of endogenous __stem cells__ [15]. These implanted __stem cells__ come from sources such as bone marrow, umbilical cord or fetal neural __stem cells__ from human adults. They are a popular choice as they reduce immunoreaction and increases the effectiveness of the therapy [3]. __Stem cells__ __improve__ brain plasticity and __repair__ by neuronal regeneration through axonal transport, the secretion of growth promoting factors such as brain derived neurotrophic factor (BDNF), remyelination and reduction of inflammation [1]. Developments in animal models and imaging methods for stroke are also important in study design considerations for human __clinical trials__. Advanced __clinical trials__ involving the use of increased doses of brain stem cells in stroke therapy have started and thus far, there have been no adverse effects. Thus far, stem cell transplantation has demonstrated to be an effective therapy for stroke in the future.

 Figure 1: BDNF modified NSCs display promoted neurite outgrowth compared to control.

** 1.1 Limitations of Current Therapy **
The long-term outcomes for stroke patients have significantly improved with the advent of more effective __rehabilitation__ programs. However, strokes continue to take a large toll on patients, the __economy__ and society, and many stroke patients remain dependent and __still__ suffer from severe neurological deficits for weeks to months after the onset of their conditions. Currently, stroke treatment centres on reducing the amount of ischemic damage and reducing the __number__ of cells that die subsequent to the stroke [15]. Unfortunately, thrombolytic agents have a __very__ narrow window of therapeutic opportunity [15].

=**2. Different stem cell therapies for stroke **= There are two strategies for stem cell therapy. First, exogenous cells can be transplanted into the ischemic infarct cortex to replace dead cells and support the remaining cells [1]. __Second__, the proliferation, differentiation, and migration of endogenous stem or progenitor cells may be enhanced [1]. Stroke models have been tested with the following cell types:

ESCs come from the inner cell mass of the pre-implantation blastocyte and are self-renewing, multipotent cells [12]. Undifferentiated ES cells are able to proliferate and fully differentiate into dopaminergic neurons. However, because of the potential risk for teratoma formation, the transplantation of undifferentiated ES cells in to stroke-afflicted animals has not been extensively examined. The __high__ cell density and species barrier __between__ donor and host cells __both__ seem to be important factors in the formation of tumours in transplanted cells [12]. The ES cells can be differentiated in vitro before implantation as a possible solution [9]. Stem cells can be taken from an adult animal, differentiated in vitro, and transplanted back into the stroke-afflicted animal [15].

NSCs are cells that keep the capacity to renew themselves and differentiate into different types of cells in the CNS. They may be __found__ in the subventricular __zone__ (SVZ) and subgranular zone (SGZ) of the brain. They are found in tissues of a fetus, neonatal, young or adult animal. NSCs have been taken from both SVZ and SGZ and successfully transplanted in animal stroke models. This resulted in differentiation, migration to the ischemic area, and improvements in behavioural __tests__. In addition, NSCs isolated from the adult brain are able to be genetically modified to only __express__ certain genes that may facilitate the regeneration process [12]. The importance of combination therapy with transplantation and rehabilitation is emphasized because it has been observed that an enriched environment improves NSC migration and functional recovery.

Figure 2: (A) Proliferation of NSC at SGZ, migration into cell layer and differentiation in dentate gyrus. (B) Proliferation of NSC at SVZ, migration towards lesion, differentiation into mature neurons.

NSCs may also be taken from induced pluripotent stem cells (iPSCs). These cells behave the same as embryonic stem cells in vitro, but are created from adult cells. They can be taken from the stroke patient himself, avoiding ethical concerns and the requirement of immunosuppression. While transplanted cells taken from iPSCs survive in the stroke patient’s brain, they are found not to reduce the stroke area or improve behavioural recovery in the month subsequent to the transplant [10]. A promising alternative source for cell replacement therapy for stroke victims comes from <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">adult autologous stem cells derived from bone marrow (BMSCs). Within the bone marrow are mesenchymal stem cells that can differentiate into osteoblasts, chondroblasts, adpocytes, and skeletal muscle [12]. The injection of MSCs into the infarct border zone of animals has been shown to lead to the regeneration of neurons, with improved survival rates and brain function. The MSCs can be injected into the carotid artery or through intravenous administration. They have the ability to penetrate the blood-brain barrier, migrate to the ischemic area, and improve neurological recovery in animal models.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Another source of autologous stem cells is menstrual blood [6]. Menstrual blood is a rich source of stromal cells, is efficient, noncontroversial, and has the ability to secrete growth factors. Stroke induced behavioural and histological deficits were improved by transplanting the menstrual blood-derived stem cells either directly into the brain or peripherally. Menstrual blood is a much more convenient source of stem cells, with a wider range of harvest time, than bone marrow or umbilical cord blood [6].

**<span style="font-family: 'Times New Roman','serif'; font-size: 16px;"> 2.1 Use of Human Neural Progenitor Stem Cells **
<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Jensen et al (2011) first reported transplanting NSCs derived from human iPSCs in rat stroke models. It is currently unclear what the optimal route of transplantation may be. There are multiple reasons for the lack of functional improvement. These include not knowing the optimal timing of transplantation after the stroke, the optimal dose of cells, or the many variables of the cells themselves. The variations between the cells themselves include variations in their source, culture protocol, and differentiation stage. Human pluripotent stem cell-derived neural progenitors may also require months of maturation before they are able to contribute to functional improvement. Therefore, while the cell transplantation approach for the treatment of stroke appears feasible, the numerous variables involved require additional study.

=**<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">3. Stem Cell Therapy in Animal Models of Stroke **= <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">While NSC therapy is now in the advanced stages of human clinical trials [14], most studies to this point have been on animals. There are several reasons that mice are generally used. As mammals, they share many homologous features and a similar brain with humans. This is useful when the studies are transplanted into clinical trials. The most often used family of mice are the Murines. The murine genome has already been sequenced and can therefore be easily correlated to the human homologue.

**<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">3.1 Imaging Methods in Stem Ce ****<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">ll Studies **
<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Cellular imaging, both //in vitro// and //in vivo//, is critical for the success of stem cell therapy for stroke [8]. The main method used to image transplants is the magnetic resonance imaging (MRI) approach, in combination with bioluminescent imaging (BLI). MRIs provides high spatial resolution to identify the stroke area and transplant, ensures success of cell delivery to desired site, and to track interactions of the transplanted cells with its surroundings [16]. Green fluorescence protein (GFP), double fusion protein (fLuc) and firefly luciferase reporter genes are needed for BLI and immunocytochemistry [8].



<span style="color: #000000; font-family: 'Times New Roman','serif'; font-size: 16px;">Figure 3: Generation of human neural stem cells (NSCs) genetically engineered to express the fLuc and GFP proteins.

=**<span style="font-family: 'Times New Roman','serif'; font-size: 16px;"> 4. Study design considerations for human clinical trials **= <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">There are issues and confounds with transferring animal model data to human clinical trials, and therefore has to be planned appropriately. A prerequisite for the advancement of clinical trials is the expectation of benefits and low risks to participants [11]. We need to take into account patient population, their natural history and prognosis as well. Stem cells used, control groups and initial safety studies need to be considered. The majority of NSC studies have used intraparenchymal delivery of cells [2] instead of intravascular administration. Delivery should be done during the first weeks after stroke, and combined with intensive physical therapies. Also, fMRI provides insights into biomarkers, which may respond to a specific intervention, and gives information about a patient’s progress [7]. Lastly, clinical trial endpoints or outcome measures need to be set within a reasonable timeline, especially one that is suitable for statistical analysis [11].

=**<span style="font-family: 'Times New Roman','serif'; font-size: 16px;"> 5. Stem cell therapy in current human studies **= <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">NSCs taken from a human teratocarinomacell line underwent two small clinical trials after they showed improved behavioral outcomes in rats in 1998 [5]. No adverse events were reported in the first human safety study, where twelve patients who suffered ischemic stroke received two doses of cells by direct injection into the basal ganglia [13]. Subsequently, Kondziolka et al. (2000) reported on a phase II efficacy trial that also had no adverse reactions reported. Current preclinical and clinical studies show that stem cell-based therapies have the potential to improve clinical outcomes in stroke patients [4]. Stem cell therapy in stroke has since come a long way, with current clinical trials on humans entering its advanced stages, with higher stem cell dose usage [1].

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">**See Also** <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">-Speech given by Gary Steinberg M.D., Ph.D., on an overview of Stem Cell Therapy for Stroke, at a CIRM meeting at Stanford University.


 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">References **

<span style="color: #000000; font-family: 'Times New Roman','serif'; font-size: 16px;">[1] Andres R. H., Horie N., Slikker W., Keren-Gill H., Ke Z., Sun G., Manley N.C., Pereira M. P., Sheikh L. A., McMillan E. L., Schaar B. T., Svendsen C. N., Bliss T. M., Steinberg G. K. Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain. Brain. (2011) **134**: 1777-1789.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[2] Bacigaluppi M., Pluchino S., Martino G. Neurological stem/precursor cells for the treatment of ischemic stroke. Journal of Neurological Science. (2008) **265**: 73–77

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[3] Barkho B. Z., Zhao X. Adult <span class="hithilite" style="font-family: 'Times New Roman','serif'; font-size: 16px;"> neural stem cells <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">: response to <span class="hithilite" style="font-family: 'Times New Roman','serif'; font-size: 16px;">stroke <span style="font-family: 'Times New Roman','serif'; font-size: 16px;"> injury and <span class="hithilite" style="font-family: 'Times New Roman','serif'; font-size: 16px;">potential <span style="font-family: 'Times New Roman','serif'; font-size: 16px;"> for therapeutic applications. Current Stem Cell Research and Therapy. (2011) **6(4)**: 327-338

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[4] Bjorkland A. Cell replacement strategies for neurodegenerative disorders. Novartis Foundation Symposium. (2000) **231**: 7-15

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[5] Borlongan C.V., Tajima Y., Trojanowski J.Q. Transplantation of cryopreserved human embryonal-carcinoma-derived neuros (NT2N) promotes functional recovery in ischemic rats. Experimental Neurology. (1998) **149**: 310–321.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[6] Borlongan C.V., Kaneko Y., Maki M., Yu S. J., Ali M., Allickson J. G., Sanberg C. D., Kuzmin-Nichols N., Sanberg P. R. Menstrual blood cells display stem cell-like phenotypic markers and exert neuroprotection following transplantion in experimental stroke. Stem Cells and Development. (2010) **19 (4)**: 439-451

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[7] Collins J.M. Functional imaging in Phase I studies: decorations or decision making? Journal of Clinical Oncology. (2003) **21**: 2807–2809.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[8] Daadi M. M., Li Z., Arac A., Grueter B. A., Sofilos M., Malenka R. C., Wu J. C., Steinberg G. K. Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain. The American Society of Gene Therapy. (2009) **17(7)**: 1282-1291

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[9] Erdo F., Buhrle C., Blunk J., Hoehn M., Xia Y., Fleischmann B., Focking M., Kustermann E., Kolossov E., Hescheler J. Host-dependent tumorigenesis of embryonic stem cell transplantation in experimental stroke. Journal of Cerebral Blood Flow and Metabolism. (2003) **23**:780–785

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[10] Jensen M. B., Yan H. M., Krishnaney-Davidson R., Al Sawaf A., Zhang S.C. Survival and differentiation of transplanted neural stem cells derived from human induced pluripotent stem cells in a rat stroke model. Journal of Stroke and Cerebrovascular Diseases. (2011) 1-5.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[11] Kalladka D., Muir K. M. Stem cell therapy in stroke: designing clinical trials. Neurochemistry International. (2011) **59**: 367-370

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[12] Kameda M., Shingo T., Takahashi K., Muraoka K., Kurozumi K., Yasuhara T., Maruo T., Tsuboi T., Uozumi T., Matsui T. Adult neural stem and progenitor cells modified to secrete GDNF can protect, migrate and integrate after intracerebral transplantation in rats with transient forebrain ischemia. European Journal of Neuroscience. (2011) **26**:1462–1478

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[13] Kondziolka D., Wechsler L., Goldstein S. Transplantation of cultured human neuronal cells for patients with stroke. Neurology. (2000) **55**: 565–569

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[14] Lin Y. C., Ko T. L., Shih Y. H., Lin M. Y., Fu T. W., Hsiao H. S., Hsu J. C., Fu Y. S. Human umbilical mesenchymal stem cells promote recovery after ischemic stroke. Stroke. (2011) **42**: 2045-2053.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[15] Luo Y. Cell-based therapy for stroke. Journal of Neural Transmission. (2011) **118**: 61-74

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">[16] van der Bogt K. E., Swijnenburg R. J., Cao F. and Wu J. C. Molecular imaging of human embryonic stem cells: keeping an eye on differentiation, tumorigenicity and <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">immunogenicity. Cell Cycle. (2006) **5**: 2748–2752.