TDO2-augmented fibroblasts secrete EVs enriched in immunomodulatory Y-derived small RNA.

Mounting evidence implicates extracellular vesicles (EVs) factors as mediators of cell therapy. Cardiosphere-derived cells are cardiac-derived cells with tissue reparative capacity. Activation of a downstream target of wnt/ß-catenin signalling, tryptophan 2,3 dioxygenase (TDO2) renders therapeutically inert skin fibroblasts cardioprotective. Here, we investigate the mechanism by which concentrated conditioned media from TDO2-augmented fibroblasts (TDO2-CCM) exert cardioprotective effects. TDO2-CCM is cardioprotective in a mouse model of MI compared to CCM from regular fibroblasts (HDF-CCM). Transcriptomic analysis of cardiac tissue at 24 h demonstrates broad suppression of inflammatory and cell stress markers in animals given TDO2-CCM compared to HDF-CCM or vehicle. Sequencing analysis of TDO2-EV RNA demonstrated abundance of a small Y-derived small RNA dubbed 'NT4'. Purification of TDO2-EVs by size-exclusion chromatography and RNAse protection assays demonstrated that NT4 is encapsulated inside EVs. Consistently with TDO2-CCM, macrophages exposed to NT4 showed suppression of the inflammatory and cell stress mediators, particularly p21/cdkn1a. NT4-depleted TDO2-CCM resulted in diminished immunomodulatory capacity. Finally, administration of NT4 alone was cardioprotective in an acute model of myocardial infarction. Taken together, these findings elucidate the mechanism by which TDO2 augmentation mediates potency in secreted EVs through enrichment of NT4 which suppresses upstream cell stress mediators including p21/cdkn1a.

Extracellular Vesicles Secreted by TDO2-Augmented Fibroblasts Regulate Pro-inflammatory Response in Macrophages.

Extracellular vesicles (EVs) are secreted lipid bilayer vesicles that mediate cell to cell communication and are effectors of cell therapy. Previous work has shown that canonical Wnt signaling is necessary for cell and EV therapeutic potency. Tryptophan 2,3-dioxygenase (TDO2) is a target gene of canonical Wnt signaling. Augmenting TDO2 in therapeutically inert fibroblasts endows their EVs with immunomodulatory capacity including attenuating inflammatory signaling in macrophages. Transcriptomic analysis showed that macrophages treated with EVs from fibroblasts overexpressing TDO2 had blunted inflammatory response compared to control fibroblast EVs. In vivo, EVs from TDO2-overexpressing fibroblasts preserved cardiac function. Taken together, these results describe the role of a major canonical Wnt-target gene (TDO2) in driving the therapeutic potency of cells and their EVs.

Engineered Fibroblast Extracellular Vesicles Attenuate Pulmonary Inflammation and Fibrosis in Bleomycin-Induced Lung Injury.

Pulmonary fibrosis is a progressive disease for which no curative treatment exists. We have previously engineered dermal fibroblasts to produce extracellular vesicles with tissue reparative properties dubbed activated specialized tissue effector extracellular vesicles (ASTEX). Here, we investigate the therapeutic utility of ASTEX in vitro and in a mouse model of bleomycin-induced lung injury. RNA sequencing demonstrates that ASTEX are enriched in micro-RNAs (miRs) cargo compared with EVs from untransduced dermal fibroblast EVs (DF-EVs). Treating primary macrophages with ASTEX reduced interleukin (IL)6 expression and increased IL10 expression compared with DF-EV-exposed macrophages. Furthermore, exposure of human lung fibroblasts or vascular endothelial cells to ASTEX reduced expression of smooth muscle actin, a hallmark of myofibroblast differentiation (respectively). In vivo, intratracheal administration of ASTEX in naïve healthy mice demonstrated a favorable safety profile with no changes in body weight, lung weight to body weight, fibrotic burden, or histological score 3 weeks postexposure. In an acute phase (short-term) bleomycin model of lung injury, ASTEX reduced lung weight to body weight, IL6 expression, and circulating monocytes. In a long-term setting, ASTEX improved survival and reduced fibrotic content in lung tissue. These results suggest potential immunomodulatory and antifibrotic properties of ASTEX in lung injury.

Small molecule inhibitors and culture conditions enhance therapeutic cell and EV potency via activation of beta-catenin and suppression of THY1.

Primary cell therapy continues to face significant hurdles to therapeutic translation including the inherent variations that exist from donor to donor, batch to batch, and scale-up driven modifications to the manufacturing process. Cardiosphere-derived cells (CDCs) are stromal/progenitor cells with clinically demonstrated tissue reparative capabilities. Mechanistic investigations have identified canonical Wnt/ß-catenin signaling as a therapeutic potency marker, and THY1 (CD90) expression as inversely correlated with potency. Here we demonstrate that the cardiosphere formation process increases ß-catenin levels and enriches for therapeutic miR content in the extracellular vesicles of these cells, namely miR-146a and miR-22. We further find that loss of potency is correlated with impaired cardiosphere formation. Finally, our data show that small GSK3ß inhibitors including CHIR, and BIO and "pro-canonical Wnt" culturing conditions can rescue ß-catenin signaling and reduce CD90 expression. These findings identify strategies that could be used to maintain CDC potency and therapeutic consistency.

A comprehensive method for identification of suitable reference genes in extracellular vesicles.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, economical and widely used methods for evaluating gene expression. However, the utility of this method continues to be undermined by a number of challenges including normalization using appropriate reference genes. The need to develop tailored and effective strategies is further underscored by the burgeoning field of extracellular vesicle (EV) biology. EVs contain unique signatures of small RNAs including microRNAs (miRs). In this study we develop and validate a comprehensive strategy for identifying highly stable reference genes in a therapeutically relevant cell type, cardiosphere-derived cells. Data were analysed using the four major approaches for reference gene evaluation: NormFinder, GeNorm, BestKeeper and the Delta Ct method. The weighted geometric mean of all of these methods was obtained for the final ranking. Analysis of RNA sequencing identified miR-101-3p, miR-23a-3p and a previously identified EV reference gene, miR-26a-5p. Analysis of a chip-based method (NanoString) identified miR-23a, miR-217 and miR-379 as stable candidates. RT-qPCR validation revealed that the mean of miR-23a-3p, miR-101-3p and miR-26a-5p was the most stable normalization strategy. Here, we demonstrate that a comprehensive approach of a diverse data set of conditions using multiple algorithms reliably identifies stable reference genes which will increase the utility of gene expression evaluation of therapeutically relevant EVs.

Exosomes as critical agents of cardiac regeneration triggered by cell therapy.

The CADUCEUS trial of cardiosphere-derived cells (CDCs) has shown that it may be possible to regenerate injured heart muscle previously thought to be permanently scarred. The mechanisms of benefit are known to be indirect, but the mediators have yet to be identified. Here we pinpoint exosomes secreted by human CDCs as critical agents of regeneration and cardioprotection. CDC exosomes inhibit apoptosis and promote proliferation of cardiomyocytes, while enhancing angiogenesis. Injection of exosomes into injured mouse hearts recapitulates the regenerative and functional effects produced by CDC transplantation, whereas inhibition of exosome production by CDCs blocks those benefits. CDC exosomes contain a distinctive complement of microRNAs, with particular enrichment of miR-146a. Selective administration of a miR-146a mimic reproduces some (but not all) of the benefits of CDC exosomes. The findings identify exosomes as key mediators of CDC-induced regeneration, while highlighting the potential utility of exosomes as cell-free therapeutic candidates.