Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell‐related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV‐based therapeutics at the preclinical and clinical levels. This review outlines topic‐specific action items that, if addressed, will enhance the development of best‐practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730–1739

Extracellular vesicles, in particular the subclass exosomes, are rapidly emerging as a novel therapeutic platform. However, currently very few clinical validation studies and no clearly defined manufacturing process exist. As exosomes progress towards the clinic for treatment of a vast array of diseases, it is important to define the engineering basis for their manufacture early in the development cycle to ensure they can be produced cost-effectively at the appropriate scale. We hypothesize that transitioning to defined manufacturing platforms will increase consistency of the exosome product and improve their clinical advancement as a new therapeutic tool. We present manufacturing technologies and strategies that are being implemented and consider their application for the transition from bench-scale to clinical production of exosomes.

Inward rectifying potassium channels facilitate cell-to-cell communication in hamster retractor muscle feed arteries.

This study sought to define whether inward rectifying K(+) (K(IR)) channels were modulated by vasoactive stimuli known to depolarize and constrict intact cerebral arteries. Using pressure myography and patch-clamp electrophysiology, initial experiments revealed a Ba(2+)-sensitive K(IR) current in cerebral arterial smooth muscle cells that was active over a physiological range of membrane potentials and whose inhibition led to arterial depolarization and constriction. Real-time PCR, Western blot, and immunohistochemical analyses established the expression of both K(IR)2.1 and K(IR)2.2 in cerebral arterial smooth muscle cells. Vasoconstrictor agonists known to depolarize and constrict rat cerebral arteries, including uridine triphosphate, U46619, and 5-HT, had no discernable effect on whole cell K(IR) activity. Control experiments confirmed that vasoconstrictor agonists could inhibit the voltage-dependent delayed rectifier K(+) (K(DR)) current. In contrast to these observations, a hyposmotic challenge that activates mechanosensitive ion channels elicited a rapid and sustained inhibition of the K(IR) but not the K(DR) current. The hyposmotic-induced inhibition of K(IR) was 1) mimicked by phorbol-12-myristate-13-acetate, a PKC agonist; and 2) inhibited by calphostin C, a PKC inhibitor. These findings suggest that, by modulating PKC, mechanical stimuli can regulate K(IR) activity and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechanoregulation of K(IR) channels plays a role in the development of myogenic tone.

Elevation of the intracellular free Ca 2؉ concentration regulates many functional responses in airway smooth muscle, including contraction, proliferation, adhesion, and cell survival. This increase in calcium can be achieved by a release from internal stores (sarcoplasmic reticulum) and/or entry across the cell membrane from the extracellular environment. The molecular identity of this calcium influx pathway in human airway smooth muscle (HASM) remains unclear. Functional studies using Fluo 4-loaded HASM suggest the presence of a histamine H 1 receptor-activated Ca 2؉ entry pathway with characteristics similar to those seen with transient receptor potential (TRP) family homologs. Using a range of molecular and cell biological approaches we defined the expression pattern of transient receptor potential classics (TRPC) homologs in airway cells and tissue. Here we show that HASM and human bronchial epithelial cells both express TRPC1,-4, and-6, with HASM also expressing TRPC3 at the mRNA level. Identification of TRPC6 protein by western blot and confocal microscopy indicated that the protein is localized in specific cell types, suggesting that it plays an important role in regulating key functions in airway cells. These data demonstrate the expression of a range of TRPC homologs in the airway and the presence of a functional Ca 2؉ entry pathway with characteristics typical of TRPC family members. TRPC homologs may provide an important novel target for the treatment of airway disease. Calcium homeostasis plays a key role in the regulation of many functional responses in airway cells. In airway smooth muscle (ASM), elevation of intracellular Ca 2ϩ levels promotes contraction and plays a role in cell proliferation, adhesion, and survival (1). The source of Ca 2ϩ for these responses has become clear over recent years. The initial contractile response of ASM is dependent upon the release of Ca 2ϩ from intracellular stores as a consequence of receptor-mediated inositol 1,4,5 triphosphate (IP 3) formation (2). However, the sustained elevation of intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i), which occurs in response to stimulation with agonists such as bradykinin and histamine,

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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...

Background: The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycER TAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycER TAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycER TAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycER TAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycER TAM transcription by qPCR and qRT-PCR, respectively.