Concise Review: Increasing the Validity of Cerebrovascular Disease Models and Experimental Methods for Translational Stem Cell Research.

Interspecies differences, anatomical and physiological aspects, as wells as simplified study designs contribute to an overestimation of treatment effects and limit the transferability of experimental results into clinical applications. Confounders of cell therapies for cerebrovascular disorders (CVD) include common CVD comorbidities, frequent medications potentially affecting endogenous and transplanted stem cells, as well as age- and immune-system-related effects. All those can contribute to a substantial modeling bias, ultimately limiting the prospective quality of preclinical research programs regarding the clinical value of a particular cell therapy. In this review, we discuss the nature and impact of most relevant confounders. We provide suggestions on how they can be considered to enhance the validity of CVD models in stem cell research. Acknowledging substantial and sometimes surprising effects of housing conditions, chronobiology, and intersex differences will further augment the translational value of animal models. We finally discuss options for the implementation of high-quality functional and imaging readout protocols. Altogether, this might help to gain a more holistic picture about the therapeutic impact of a particular cell therapy for CVD, but also on potential side and off-site effects of the intervention. Stem Cells 2017;35:1141-1153.

Concise Review: MSC Adhesion Cascade-Insights into Homing and Transendothelial Migration.

Mesenchymal stem cells (MSCs) are promising candidates for adult cell therapies in regenerative medicine. To fully exert their potential, efficient homing and migration toward lesion sites play an important role. Local transplantation deposits MSC in spatial proximity to the lesion, but often requires invasive procedures. Systemic administration routes are favored, but require the targeted extravasation of the circulating MSC at the site of injury. Transplanted MSC can indeed leave the blood flow and transmigrate through the endothelial barrier, and reach the lesion site. However, the underlying processes are not completely dissolved yet. Recent in vitro and in vivo research identified some key molecules scattered light on the extravasation mechanism. This review provides a detailed overview over the current knowledge of MSC transendothelial migration. We use the leukocyte extravasation process as a role model to build a comprehensive concept of MSC egress mechanisms from the blood stream and identified relevant similarities as well as important differences between the extravasation mechanisms. Stem Cells 2017;35:1446-1460.

Integrin α4 Overexpression on Rat Mesenchymal Stem Cells Enhances Transmigration and Reduces Cerebral Embolism After Intracarotid Injection.

BACKGROUND AND PURPOSE: Very late antigen-4 (integrin α4ß1)/vascular cell adhesion molecule-1 mediates leukocyte trafficking and transendothelial migration after stroke. Mesenchymal stem cells (MSCs) typically express integrin ß1 but insufficient ITGA4 (integrin α4), which limits their homing after intravascular transplantation. We tested whether ITGA4 overexpression on MSCs increases cerebral homing after intracarotid transplantation and reduces MSC-borne cerebral embolism. METHODS: Rat MSCs were lentivirally transduced to overexpress ITGA4. In vitro transendothelial migration was assessed using a Boyden chamber assay. Male Wistar rats intracarotidly received 0.5×106 control or modified MSCs 24 hours after sham or stroke surgery. In vivo behavior of MSCs in the cerebral vasculature was observed by intravital microscopy and single-photon emission computed tomography for up to 72 hours. RESULTS: Transendothelial migration of ITGA4-overexpressing MSCs was increased in vitro. MSCs were passively entrapped in microvessels in vivo and occasionally formed large cell aggregates causing local blood flow interruptions. MSCs were rarely found in perivascular niches or parenchyma at 72 hours post-transplantation, but ITGA4 overexpression significantly decreased cell aggregation and ameliorated the evoked cerebral embolism in stroke rats. CONCLUSIONS: ITGA4 overexpression on MSCs enhances transendothelial migration in vitro, but not in vivo, although it improves safety after intracarotid transplantation into stroke rats.

Physical therapy exerts sub-additive and suppressive effects on intracerebral neural stem cell implantation in a rat model of stroke.

Intracerebral cell therapy (CT) is emerging as a new therapeutic paradigm for stroke. However, the impact of physical therapy (PT) on implanted cells and their ability to promote recovery remains poorly understood. To address this translational issue, a clinical-grade neural stem cell (NSC) line was implanted into peri-infarct tissue using MRI-defined injection sites, two weeks after stroke. PT in the form of aerobic exercise (AE) was administered 5 × per week post-implantation using a paradigm commonly applied in patients with stroke. A combined AE and CT exerted sub-additive therapeutic effects on sensory neglect, whereas AE suppressed CT effects on motor integration and grip strength. Behavioral testing emerged as a potentially major component for task integration. It is expected that this study will guide and inform the incorporation of PT in the design of clinical trials evaluating intraparenchymal NSCs implantation for stroke.

ECM hydrogel improves the delivery of PEG microsphere-encapsulated neural stem cells and endothelial cells into tissue cavities caused by stroke.

Intracerebral implantation of neural stem cells (NSCs) to treat stroke remains an inefficient process with <5% of injected cells being retained. To improve the retention and distribution of NSCs after a stroke, we investigated the utility of NSCs' encapsulation in polyethylene glycol (PEG) microspheres. We first characterized the impact of the physical properties of different syringes and needles, as well as ejection speed, upon delivery of microspheres to the stroke injured rat brain. A 20 G needle size at a 10 µL/min flow rate achieved the most efficient microsphere ejection. Secondly, we optimized the delivery vehicles for in vivo implantation of PEG microspheres. The suspension of microspheres in extracellular matrix (ECM) hydrogel showed superior retention and distribution in a cortical stroke caused by photothrombosis, as well as in a striatal and cortical cavity ensuing middle cerebral artery occlusion (MCAo). Thirdly, NSCs or NSCs + endothelial cells (ECs) encapsulated into biodegradable microspheres were implanted into a large stroke cavity. Cells in microspheres exhibited a high viability, survived freezing and transport. Implantation of 110 cells/microsphere suspended in ECM hydrogel produced a highly efficient delivery that resulted in the widespread distribution of NSCs in the tissue cavity and damaged peri-infarct tissues. Co-delivery of ECs enhanced the in vivo survival and distribution of ∼1.1 million NSCs. The delivery of NSCs and ECs can be dramatically improved using microsphere encapsulation combined with suspension in ECM hydrogel. These biomaterial innovations are essential to advance clinical efforts to improve the treatment of stroke using intracerebral cell therapy.

Translation, but not transfection limits clinically relevant, exogenous mRNA based induction of alpha-4 integrin expression on human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) represent promising resource of cells for regenerative medicine in neurological disorders. However, efficient and minimally invasive methods of MSCs delivery to the brain still have to be developed. Intra-arterial route is very promising, but MSCs are missing machinery for diapedesis through blood-brain barrier. Thus, here we have tested a mRNA-based method to induce transient expression of ITGA4, an adhesion molecule actively involved in cell extravasation. We observed that transfection with an ITGA4-mRNA construct bearing a conventional cap analogue (7-methylguanosine) failed to produce ITGA4 protein, but exogenous ITGA4-mRNA was detected in transfected MSCs. This indicates that not transfection, but rather translation being the major roadblock. Stabilization of ITGA4-mRNA with SSB proteins resulted in ITGA4 protein synthesis in HEK293 cells only, whereas in MSCs, satisfactory results were obtained only after using an anti-reverse-cap-analogue (ARCA). The presence of ITGA4 protein in MSCs was transient and lasted for up to 24 h after transfection. Membranous location was confirmed by flow cytometry of viable non-permeabilized cells using anti-ITGA4 antibody. The mRNA-based expression of itga4 transgene is potentially sufficient for diapedesis after intra-arterial delivery. To conclude, mRNA-based engineering of stem cells is a rapid and integration-free method and attractive from the perspective of potential future clinical application.

Combined enriched environment÷atipamezole treatment transiently improves sensory functions in stroke rats independent from neurogenesis and angiogenesis.

Functional recovery after cerebral ischemia may be enhanced by activation of the noradrenergic system and by environmental enrichment. The underlying mechanisms have remained elusive, but endogenous neurogenesis and perilesional angiogenesis have been speculated to contribute to the behavioral improvement. To address this question, neurogenesis in the subventricular zone (SVZ) and perilesional angiogenesis (RECA-1) were correlated with behavioral performance in forty Wistar rats subjected to transient middle cerebral artery occlusion (MCAO) or sham-operation. Atipamezole, an α2-adrenoreceptor antagonist (1 mg÷kg, i.p.), was administered for 10 days together with housing of rats in an enriched environment. MCAO rats and sham-operated rats housed in single non-enriched cages were used as controls. Histological analysis after 28-day behavioral follow-up showed a massive increase in doublecortin (DCX)-positive cells in the SVZ both in MCAO rats housed in single cages and in the enriched environment together with atipamezole treatment whereas perilesional RECA-1 staining for new blood vessels was not altered. Time to the first contact and time to remove sticky stimuli from the forelimbs indicated improved sensory processing, which disappeared after cessation of atipamezole administration. Skilled forelimb use as measured by performance in Montoya's staircase test was not affected by the treatment. There were no correlations between behavioral measures and histology. Thus, sensory facilitation or reversal of hypometabolism by the combined therapy may be the mechanism accounting for the improved behavior after stroke independent from neurogenesis and angiogenesis.

The cerebral embolism evoked by intra-arterial delivery of allogeneic bone marrow mesenchymal stem cells in rats is related to cell dose and infusion velocity.

INTRODUCTION: Intra-arterial cell infusion is an efficient delivery route with which to target organs such as the ischemic brain. However, adverse events including microembolisms and decreased cerebral blood flow were recently reported after intra-arterial cell delivery in rodent models, raising safety concerns. We tested the hypothesis that cell dose, infusion volume, and velocity would be related to the severity of complications after intra-arterial cell delivery. METHODS: In this study, 38 rats were subjected to a sham middle cerebral artery occlusion (sham-MCAO) procedure before being infused with allogeneic bone-marrow mesenchymal stem cells at different cell doses (0 to 1.0 × 10(6)), infusion volumes (0.5 to 1.0 ml), and infusion times (3 to 6 minutes). An additional group (n = 4) was infused with 1.0 × 10(6) cells labeled with iron oxide for in vivo tracking of cells. Cells were infused through the external carotid artery under laser Doppler flowmetry monitoring 48 hours after sham-MCAO. Magnetic resonance imaging (MRI) was performed 24 hours after cell infusion to reveal cerebral embolisms or hemorrhage. Limb placing, cylinder, and open field tests were conducted to assess sensorimotor functions before the rats were perfused for histology. RESULTS: A cell dose-related reduction in cerebral blood flow was noted, as well as an increase in embolic events and concomitant lesion size, and sensorimotor impairment. In addition, a low infusion velocity (0.5 ml/6 minutes) was associated with high rate of complications. Lesions on MRI were confirmed with histology and corresponded to necrotic cell loss and blood-brain barrier leakage. CONCLUSIONS: Particularly cell dose but also infusion velocity contribute to complications encountered after intra-arterial cell transplantation. This should be considered before planning efficacy studies in rats and, potentially, in patients with stroke.

Human bone marrow mesenchymal stem/stromal cells produce efficient localization in the brain and enhanced angiogenesis after intra-arterial delivery in rats with cerebral ischemia, but this is not translated to behavioral recovery.

Intravascular cell therapy is a promising approach for the treatment of stroke. However, high accumulation of cells to lungs and other filtering organs is a major concern after intravenous (i.v.) cell transplantation. This can be circumvented by intra-arterial (i.a.) cell infusion, which improves homing of cells to the injured brain. We studied the effect of i.a. delivery of human bone marrow-derived mesenchymal cells (BMMSCs) on behavioral and histological outcome in rats after middle cerebral artery occlusion (MCAO). Sixty male Wistar rats were subjected to transient MCAO (60 min) or sham-operation. BMMSCs (1×10(6)) were infused into the external carotid artery on postoperative day 2 or 7. Histology performed after a 42-day follow-up did not detect any human cells (MAB1281) in the ischemic brain. Endothelial cell staining with RECA-1 revealed a significant increase in the number of blood vessels in the perilesional cortex in MCAO rats treated with cells on postoperative day 7. Behavioral recovery as assessed in three tests, sticky label, cylinder and Montoya's staircase, was not improved by human BMMSCs during the follow-up. In conclusion, human BMMSCs did not improve functional recovery in MCAO rats despite effective initial homing to the ischemic hemisphere and enhanced angiogenesis, when strict behavioral tests not affected by repeated testing and compensation were utilized.

Transplantation of placenta-derived mesenchymal stromal cells upon experimental stroke in rats.

The beneficial effects of bone marrow-derived mesenchymal stromal cell (MSC) administration following experimental stroke have already been described. Despite several promising characteristics, placenta-derived MSC have not been used in models of focal ischemia. The aim of the current study is to investigate the impact of intravenously transplanted placenta-derived MSC on post-stroke recovery. Permanent occlusion of the middle cerebral artery was induced in spontaneously hypertensive rats. MSC were obtained from the human maternal or fetal placenta and intravenously administered after 24 h (single transplantation) or after 8 h and 24 h (dual transplantation). Sensorimotor deficits were quantified for 60 days using the beam walk test and the modified Neurological Severity Score system. Infarct volume was determined in vivo by means of magnetic resonance imaging on days 1, 8, 29 and 60. Astroglial reactivity was semiquantitatively ascertained within a small and a broad region adjacent to the lesion border. The double infusion of placental MSC was superior to single transplantation in the functional tests. However, a significant difference to the control group in all outcome parameters was observed only for maternally derived MSC. These findings suggest that placental tissue constitutes a promising source for experimental stroke therapies.