Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

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.

c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain.

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-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM 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-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM 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-mycERTAM transcription by qPCR and qRT-PCR, respectively. RESULTS: The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence. CONCLUSION: In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

Implantation of c-mycER TAM immortalized human mesencephalic-derived clonal cell lines ameliorates behavior dysfunction in a rat model of Parkinson's disease.

Human neural stem cells offer the hope that a cell therapy treatment for Parkinson's disease (PD) could be made widely available. In this study, we describe two clonal human neural cell lines, derived from two different 10-week-old fetal mesencephalic tissues and immortalized with the c-mycER(TAM) transgene. Under the growth control of 4-hydroxytamoxifen, both cell lines display stable long-term growth in culture with a normal karyotype. In vitro, these nestin-positive cells are able to differentiate into tyrosine hydroxylase (TH)-positive neurons and are multipotential. Implantation of the undifferentiated cells into the 6-OHDA substantia nigral lesioned rat model displayed sustained improvements in a number of behavioral tests compared with noncell-implanted, vehicle-injected controls over the course of 6 months. Histological analysis of the brains showed survival of the implanted cells but no evidence of differentiation into TH-positive neurons. An average increase of approximately 26% in host TH immunoreactivity in the lesioned dorsal striatum was observed in the cell-treated groups compared to controls, with no difference in loss of TH cell bodies in the lesioned substantia nigra. Further analysis of the cell lines identified a number of expressed trophic factors, providing a plausible explanation for the effects observed in vivo. The exact mechanisms by which the implanted human neural cell lines provide behavioral improvements in the PD model are not completely understood; however, these findings provide evidence that cell therapy can be a potent treatment for PD acting through a mechanism independent of dopaminergic neuronal cell replacement.

The functional consequence of RhoA knockdown by RNA interference in rat cerebral arteries.

Uridine triphosphate (UTP) constricts cerebral arteries by activating transduction pathways that increase cytosolic [Ca(2+)] and myofilament Ca(2+) sensitivity. The signaling proteins that comprise these pathways remain uncertain with recent studies implicating a role for several G proteins. To start clarifying which G proteins enable UTP-induced vasoconstriction, a small interfering RNA (siRNA) approach was developed to knock down specified targets in rat cerebral arteries. siRNA directed against G(q) and RhoA was introduced into isolated cerebral arteries using reverse permeabilization. Following a defined period of organ culture, arteries were assayed for contractile function, mRNA levels, and protein expression. Targeted siRNA reduced RhoA or G(q) mRNA expression by 60-70%, which correlated with a reduction in RhoA but not G(q) protein expression. UTP-induced constriction was abolished in RhoA-depleted arteries, but this was not due to a reduction in myosin light chain phosphorylation. UTP-induced actin polymerization was attenuated in RhoA-depleted arteries, which would explain the loss of agonist-induced constriction. In summary, this study illustrates that siRNA approaches can be effectively used on intact arteries to induce targeted knockdown given that the protein turnover rate is sufficiently high. It also demonstrates that the principal role of RhoA in agonist-induced constriction is to facilitate the formation of F-actin, the physical structure to which phosphorylated myosin binds to elicit arterial constriction.

Hyposmotic challenge inhibits inward rectifying K+ channels in cerebral arterial smooth muscle cells.

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.

Biosynthesized matrix provides a key role for survival signaling in bronchial epithelial cells.

The extracellular matrix (ECM) influences a variety of cellular functions, including survival, adhesion molecule expression, differentiation, and migration. The ECM composition of the epithelial basement membrane is altered in asthmatics. In this study, we elucidate the major survival signals received by bronchial epithelial cells in vitro by studying the effects of a variety of ECM factors and soluble growth factors on bronchial epithelial cell survival. Our findings indicate that the insulin family of soluble growth factors provides important survival signals but also that adhesion to ECM is a crucial determinant of bronchial epithelial cell survival. In the BEAS-2B bronchial epithelial cell line, collagens I and IV, laminin, fibronectin, and vitronectin provide significant levels of protection from apoptosis. Tenascin-C has no effect, whereas elastin and collagen V increase apoptosis to above control levels. BEAS-2B cells secrete their own biosynthesized matrix (BSM), which also provides rescue from apoptosis. Protection by collagen I, fibronectin, and vitronectin was found to be via an RGD domain. Laminin-, collagen IV-, and BSM-mediated survival is not RGD dependent. Primary bronchial epithelial cells exhibit a similar pattern of apoptosis rescue to the BEAS-2B cell line, although we did not observe any vitronectin-mediated protection in the primary cells. These data indicate that bronchial epithelial cell survival is dependent both on soluble growth factors and on a variety of ECM-derived signals.

Expression of transient receptor potential C6 and related transient receptor potential family members in human airway smooth muscle and lung tissue.

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.