Clinical-Grade Human Neural Stem Cells Promote Reparative Neovascularization in Mouse Models of Hindlimb Ischemia

Abstract

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...

When the Vessels Use Their Brain for Therapeutic Revascularization

“…3 In an experimental model of CLI, they showed that treatment with hNSCs promoted neovascularization of the ischemic limb in immunodeficient animals as well as in immunocompetent CD-1 and streptozotocin-induced diabetic mice. 2 Comparison of the hNSCs with human mesenchymal stem cells indicated a similar improvement in blood flow. Histological studies in ischemic leg of immunocompetent mice during the first 7 days after treatment revealed short-term hNSC survival, transient elevation of early host muscle inflammatory and angiogenic responses, and acceleration of myogenesis.…”

“…Histological studies in ischemic leg of immunocompetent mice during the first 7 days after treatment revealed short-term hNSC survival, transient elevation of early host muscle inflammatory and angiogenic responses, and acceleration of myogenesis. 2 However, grafted hNSCs mainly acquired an inflammatory phenotype, and the early transient increases in inflammatory cytokines, chemokines, and growth factor expression were observed in hNSC-treated ischemic muscle although classic inflammatory cell responses were not affected. Hence, the inflammatory phenotype and function of hNSCs are most likely induced by the implantation into the ischemic environment of immunocompetent mice.…”

“…Interestingly, in this study, the immunogenic potential of hNSCs seemed to be low, and there was comparable efficacy in both nude and wild-type CD-1 mice, both at short-term and long-term, without any evidence of rejection of these cells from the recipient animals. 2 This could be explained by a previous study that showed that CTX0E03 hNSCs do not express major histocompatibility complex II and do not promote a sustained host cellular response. 3 The other well-known prototype of immunoprivileged cells suitable for allogeneic applications is human mesenchymal stem cells.…”

Vascular Regeneration by Stem/Progenitor Cells

“…61 CTX0E03, a line of human neural stem cells thought to promote angiogenesis and neurogenesis in stroke, also showed progress in a hind limb ischemia model. 62 Though mesenchymal stem cells have a therapeutic benefit, recent studies suggest that they may also play a role in the pathology of aortic valve disease. 63 One potential mechanism for this is regulation of endothelial-to-mesenchymal transitions during stem cell differentiation.…”

Stem/Progenitor Cells in Vascular Regeneration

“…64 Interestingly, a clinical-grade human neural stem cell line promotes angiogenesis and neurogenesis in a preclinical model of stroke and is now showed the therapeutic activity of intramuscular implantation in murine models of hindlimb ischemia. 65 In addition, mouse iPS cell-derived Flk-1 + or Flk-1 − cells were intravenously injected into the mice after vascular injury that significantly attenuated neointimal hyperplasia when compared with controls. 62 Administration of iPS cellderived Flk-1 + cells significantly enhanced reendothelialization when compared with that of the Flk-1 − cell control group, 62 indicating a potentially useful therapeutic means for vascular dysfunction and prevention of restenosis after angioplasty.…”