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A Therapeutic Stroke in the Right Direction?

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‘Implantation site and lesion topology determine efficacy of a human neural stem cell line in a rat model of chronic stroke’

From Stem Cells 
Commentary by Carla B. Mellough

Stroke remains one of the leading causes of mortality and adult disability in developed nations1-3. For the survivors of stroke, the resulting disability is often persistent in nature and severely affects an individual’s quality of life, with the additional possibility of recurrent stroke events a grim reality. Over the past four decades, while the incidence of stroke has declined in high-income countries there has been a greater than 100% increase in low to middle-income countries, highlighting the magnitude of this cardiovascular problem.3 Yet strategies to maximize recovery and help improve patient outcome following stroke have made little progress, with only 3% of patients currently gaining any benefit from existing treatment strategies, such as the use of thrombolytic agents.4 There is therefore an obvious need for the development of restorative therapies which would help to address the loss of neurons and glia in the brain following stroke. For a cell-based therapeutic approach, determining the optimal cell type for use that will apply to a broad cross section of stroke patients while giving consistent results and minimal complications is key. Various cell types have been tested in experimental stroke ranging from embryonic, mesenchymal and neural stem cells to human umbilical cord blood, in addition to some less obvious candidates such as adipose and menstrual blood cells, with preclinical studies indicating varying positive outcomes but, importantly, that many different cell types can elicit encouraging results.4,5 Many of the cell types that have been tested appear to exert their effects not by cell replacement, but by their neurotrophic and anti-inflammatory effects on affected tissues, and therefore act by minimizing damage and providing protection to brain tissues. Neuronal precursors derived from human embryonic stem cells (hESCs) tested in a rat model of stroke were shown to elicit some functional recovery,6 but the application of such strategies to the clinic remain somewhat hampered by the potential tumorigenicity of any residual stem cells which may be transplanted alongside more differentiated cell types. Until measures to reduce this potentiality are optimized, human neural stem cells (hNSCs) represent a good alternative. In the current study, Smith et al.7 report on the use of a human cortical neuroepithelium-derived hNSC line (CTX0E03) which, in previous studies has shown efficacy in improving sensorimotor function in a dose-dependent manner and, unlike other hNSCs, shows limited migration capacity thereby providing a locally-acting cell based therapy which makes it potentially safer than other hNSC types. This work represents a collaborative study from Kings College London, the UK based company ReNeuron and the University of Pittsburgh in Pennsylvania.

The authors used middle cerebral artery occlusion (MCAo) in rat as their experimental model of stroke, which consistently affects the striatal region but can also variably affect adjacent cortical tissue. This method gives rise to persistent deficits in sensorimotor function, consistent with chronic deficits found in human stroke patients. Importantly, using magnetic resonance imaging (MRI) techniques the authors determined the extent of stroke damage and lesion topology in each case prior to implantation, a variable that is often ignored when assessing the efficacy of different therapeutic approaches on experimental stroke outcome. For hNSC transplantation, two routes of administration were tested – intracerebroventricular (ICV), which relies on the flow of cerebrospinal fluid to the brain for cellular delivery, and intraparenchymal transplantation into the peri-infarct region, which introduces cells much closer to the affected site and into a more structured microenvironment in comparison to ICV injection, amongst a network of extracellular matrix (ECM), vasculature and dying cells alongside uncompromised tissue. The authors injected 450,000 cells into the ICV fluid or peri-infarct region 14 days after stroke insult. Sensorimotor, motor and cognitive outcome was subsequently measured by water maze, rotameter, footfault and bilateral asymmetry tests.

Behavioural dysfunction tests revealed that there were no discernable improvements in function following ICV injection, compared to the sham-injected intraparenchymal hosts. In contrast, intraparenchymal transplants resulted in a gradual improvement in sensorimotor function from 4 to 10 weeks following injection, but with no further improvements after this time. These animals also showed a significant improvement in motor coordination at 12 weeks although no improvement in learning and memory could be detected. The authors observed that animals displaying damage confined to the striatum only showed greater overall functional recovery (83% improvement) than those with both striatal and cortical damage (48%). Consistent with functional recovery results, post-mortem histological analysis revealed that ICV grafts had not survived transplantation whereas intraparenchymal grafts did, with an almost seven-fold increase in the number of grafted cells in animals with striatal plus cortical damage, compared to those with striatal damage alone. Interestingly, while behavioural function tests indicated greater improvements in animals with confined striatal damage, implanted cells gave rise to FOX3-positive cortical neurons (2%) only in animals showing striatal plus cortical damage, while a further 20% gave rise to astrocytes. In cases where damage was confined to the striatum, grafted cells gave rise to astrocytes only, with increased astrocytic production associated with a reduction in spared striatal volume following MCAo. Collagen IV expression in the basement membrane of blood vessels is an indicative marker of blood vessel integrity and is significantly reduced in the peri-infarct region following stroke. Further to its positive effect on functional recovery, intraparenchymal injection of hNSCs replenished Collagen IV back to control levels, indicating that the neurovascular niche is an important contributor to post-stroke recovery. Changes in the thickness of the subependymal zone (SEZ), a region of adult neurogenesis, were also observed which were related to lesion volume and correlated with functional test outcome, but the authors were unable to establish a direct causal effect of the SEZ on stroke outcome or functional recovery.

This work highlights the importance of implantation site and lesion topology on functional outcome following stroke, and that cell-based therapy in this instance was most efficacious in striatal-only lesions. It is interesting that only intraparenchymal transplants were able to significantly improve outcome, although the authors note that transplanting a greater number of cells into animals showing greater lesion volumes may enhance outcome in these subjects. Further, if multiple areas are affected then this might require additional site-specific injections into damaged areas in order to counteract this deficit. It is likely that a smaller, more restricted area of damage may be easier to treat than larger multiple areas of damage. In contrast to what one might expect, astrocytic differentiation of grafted cells was positively associated with functional outcome, which the authors suggest may be due to the activity of their secreted factors on neural plasticity as well as ECM production and vascular recovery. Thus it seems that neuronal replacement may not be absolutely necessary for enhanced functional recovery following stroke, although the translation of this effect in human would have to be confirmed, as well as whether the sensorimotor improvements observed in the rat model used herein would be sufficient to elicit improvements in a more complex system and, if so, how much and for how long?

 

References

1. The Stroke Association: Facts and Figures about Stroke.

2. Roger VL et al. (2011) Heart Disease and Stroke Statistics—2011 Update: A report from the American Heart Association. Circulation. 123:e18–e209.

3. Feigin VL et al. (2009) Worldwide stroke incidence and early case fatality reported in 56 population-based studies: a systematic review. The Lancet Neurology 8(4):355–369.

4. Gopurappilly R et al. (2011) Stem cells in stroke repair: current success & future prospects. CNS Neurol Disord Drug Targets. 10(6):741-56.

5. Borlongan CV et al. (2010) Menstrual blood cells display stem cell-like phenotypic markers and exert neuroprotection following transplantation in experimental stroke. Stem Cells Dev. 19(4):439-52.

6. Wei L et al.  (2005) Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia. Neurobiol Dis. 19(1-2):183-93.

7. Smith EJ et al. (2012) Implantation site and lesion topology determine efficacy of a human neural stem cell line in a rat model of chronic stroke. Stem Cells 30(4):785-96.