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Abstract-We evaluated the healing potential of human fetal aorta-derived CD133ϩ progenitor cells and their conditioned medium (CD133 ϩ CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2ϫ10 4 CD133 ϩ or CD133 Ϫ cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133 ϩ cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133Ϫ cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133ϩ cells accelerated wound closure as compared with CD133Ϫ or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133ϩ cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133ϩ CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by coadministering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133 ϩ CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133 ϩ CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. Key Words: ischemia Ⅲ wound healing Ⅲ diabetes Ⅲ stem cells Ⅲ angiogenesis C hronic wounds represent a relevant clinical and socioeconomic burden, with diabetic foot ulcers alone causing costs of 300 million pounds per annum to the United Kingdom National Health System. 1 Diabetic patients with foot ulcers associated with peripheral vascular disease manifest the worst outcome, with higher amputation and mortality rates than patients carrying nonischemic ulcers. 2,3 Although the efficacy of a topical gel formulation of recombinant human platelet-derived growth factor-BB was recently demonstrated in patients with nonischemic neuropathic ulcers, 4 most ischemic ulcers are refractory to conventional treatment and growth factor (GF) therapy. 5 Therefore, new strategies for the cure of life-threatening ischemic ulcers are urgently awaited.Preliminary evidence supports the potential of adult or fetal stem/progenitor cells for the healing of skin ulcers. 6 -8 However, because of the lack of an appropriate preclinical model, no information is available regarding the effectiveness of cell therapy on ischemic diabetic foot ulcers. The healing activity of stem cells is credited to their ability to transdifferentiate into the vascular and nonvascular components of injured tissue, as well as to secretion of GFs, which may activate endogenous modulators of angiogenesis in the recipient. 9 -11 Notably, fetal stem cells show significant advantages over their adult counterparts in terms of proliferative capa...

Stroke remains one of the most promising targets for cell therapy. Thorough preclinical efficacy testing of human neural stem cell (hNSC) lines in a rat model of stroke (transient middle cerebral artery occlusion) is, however, required for translation into a clinical setting. Magnetic resonance imaging (MRI) here confirmed stroke damage and allowed the targeted injection of 450,000 hNSCs (CTX0E03) into peri-infarct tissue, rather than the lesion cyst. Intraparenchymal cell implants improved sensorimotor dysfunctions (bilateral asymmetry test) and motor deficits (footfault test and rotameter). Importantly, analyses based on lesion topology (striatal vs. striatal 1 cortical damage) revealed a more significant improvement in animals with a stroke confined to the striatum. However, no improvement in learning and memory (water maze) was evident. An intracerebroventricular injection of cells did not result in any improvement. MRI-based lesion, striatal and cortical volumes were unchanged in treated animals compared to those with stroke that received an intraparenchymal injection of suspension vehicle. Grafted cells only survived after intraparenchymal injection with a striatal 1 cortical topology resulting in better graft survival (16,026 cells) than in animals with smaller striatal lesions (2,374 cells). Almost 20% of cells differentiated into glial fibrillary acidic protein1 astrocytes, but <2% turned into FOX31 neurons. These results indicate that CTX0E03 implants robustly recover behavioral dysfunction over a 3-month time frame and that this effect is specific to their site of implantation. Lesion topology is potentially an important factor in the recovery, with a stroke confined to the striatum showing a better outcome compared to a larger area of damage. STEM CELLS 2012;30:785-796 Disclosure of potential conflicts of interest is found at the end of this article.

Exosomes are small (30–100 nm) membrane vesicles secreted by a variety of cell types and only recently have emerged as a new avenue for cell-to-cell communication. They are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. MicroRNAs (miRNAs) are short non-coding RNAs which regulate biological processes and can be found in exosomes. Here we characterized the miRNA contents of exosomes derived from human neural stem cells (hNSCs). Our investigated hNSC line is a clonal, conditionally immortalized cell line, compliant with good manufacturing practice (GMP), and in clinical trials for stroke and critical limb ischemia in the UK (clinicaltrials.gov: NCT01151124, NCT02117635, and NCT01916369). By using next generation sequencing (NGS) technology we identified the presence of a variety of miRNAs in both exosomal and cellular preparations. Many of these miRNAs were enriched in exosomes indicating that cells specifically sort them for extracellular release. Although exosomes have been proven to contain miRNAs, the copy number quantification per exosome of a given miRNA remains unclear. Herein we quantified by real-time PCR a highly shuttled exosomal miRNA subtype (hsa-miR-1246) in order to assess its stoichiometry per exosome. Furthermore, we utilized an in vitro system to confirm its functional transfer by measuring the reduction in luciferase expression using a 3’ untranslated region dual luciferase reporter assay. In summary, NGS analysis allowed the identification of a unique set of hNSC derived exosomal miRNAs. Stoichiometry and functional transfer analysis of one of the most abundant identified miRNA, hsa-miR-1246, were measured to support biological relevance of exosomal miRNA delivery.

CTX0E03 is a human neural stem cell line previously reported to reduce sensory motor deficits in a middle cerebral artery occlusion (MCAo) model of stroke. The objective of this study was to investigate if CTX0E03 treatment promotes angiogenesis. As stroke leads to damage of the vasculature in the brain, angiogenesis may contribute to the functional recovery. To test this hypothesis, the angiogenic activity of CTX0E03 was assessed both in vitro and in vivo. In vitro, CTX0E03 expression of trophic and proangiogenic factors was determined by real-time RT-PCR, Western blot, and ELISA, and its angiogenic activity was investigated in well-established angiogenesis assays. In vivo, angiogenesis was investigated in naive mice and MCAo rat brain and was evaluated by immunohistochemistry (IHC) using Von Willebrand factor (VWF), a marker of blood vessel formation, and BrdU/CD31 double labeling in naive mice only. In vitro results showed that CTX0E03-conditioned medium and coculture significantly increased total tubule formation compared with controls (p=0.002 and p=0.0008, respectively). Furthermore, CTX0E03 cells were found to be in direct association with the tubules by ICC. In vivo CTX0E03-treated brains demonstrated a significant increase in areas occupied by VWF-positive microvessels compared with vehicle-treated naive mice (two-way ANOVA, Interaction p<0.05, Treatment p<0.0001, Time p<0.0) and MCAo rat (p=0.001 unpaired t test, Welch's correction). CTX0E03-treated naive mouse brains showed an increase in BrdU/CD31 colabeling. In conclusion, in vitro CTX0E03 cells express proangiogenic factors and may promote angiogenesis by both release of paracrine factors and direct physical interaction. Furthermore, in vivo CTX0E03-treated rodent brains exhibited a significant increase in microvessels at the site of implantation compared with vehicle-injected groups. Taken together these data suggest that CTX0E03 cell therapy may provide significant benefit to stroke patients through upregulation of angiogenesis in the ischemic brain.

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C ritical limb ischemia (CLI) and ischemic stroke are common manifestations of atherosclerosis and vascular occlusion of peripheral and cerebral arteries, respectively, leading to cell death and tissue necrosis. CLI is characterized by pain at rest, nonhealing wounds, and gangrene, progressing to loss of limb and high rates of mortality. The leading risk factors of CLI are diabetes mellitus and age. Currently, there are no effective pharmacological interventions to treat CLI. Revascularization through endovascular or surgical techniques to improve patency of the affected region is only tenable in half of the patients with CLI with the achieved patency failing in 30% of cases within 1 year.1 Approximately 50% of all CLI patients die within 1 year of diagnosis. The incidence of CLI in the Western world is ≈220 new cases per million people per year, and the population at risk is expected to increase with aging and the increase in type II diabetes mellitus. 2,3 There is clearly a need to develop new therapies to restore blood flow and rescue limbs in patients with CLI. See accompanying editorial on page 237A growing therapeutic strategy for CLI is the promotion of neovascularization either by delivery of proangiogenic factors or cell therapy. The rationale of the approach is to encourage spontaneous neovascularization, which is impaired in aged or diseased patients. 4 Gene therapies, for example, vascular endothelial growth factor (VEGF) and fibroblast growth factor, have been developed to promote neovascularization in ischemic tissues; however, phase II clinical trials did not show consistent improvements in amputation-free survival. 4 There are several possible reasons for these poor outcomes, including short half-lives of vectors and possible immune/inflammatory responses to the virus. It has also been noted that the elevated production of a single growth factor can lead to the defective structure of the newly formed capillaries. Objective-CTX0E03 (CTX) is a clinical-grade human neural stem cell (hNSC) line that promotes angiogenesis and neurogenesis in a preclinical model of stroke and is now under clinical development for stroke disability. We evaluated the therapeutic activity of intramuscular CTX hNSC implantation in murine models of hindlimb ischemia for potential translation to clinical studies in critical limb ischemia. Approach and Results-Immunodeficient (CD-1 Fox nu/nu ) mice acutely treated with hNSCs had overall significantly increased rates and magnitude of recovery of surface blood flow (laser Doppler), limb muscle perfusion (fluorescent microspheres, P<0.001), and capillary and small arteriole densities in the ischemic limb (fluorescence immunohistochemistry, both P<0.001) when compared with the vehicle-treated group. Hemodynamic and anatomic improvements were dose related and optimal at a minimum dose of 3×10 5 cells. Dose-dependent improvements in blood flow and increased vessel densities by hNSC administration early after ischemia were confirmed in immunocompetent CD-1 and streptozotocin-indu...