Potency Assays for Mesenchymal Stromal Cell Secretome-Based Products for Tissue Regeneration.

Adult stem cells maintaining tissue homeostasis and regeneration are tightly regulated by their specific microenvironments or stem cell niches. The dysfunction of niche components may alter the activity of stem cells and ultimately lead to intractable chronic or acute disorders. To overcome this dysfunction, niche-targeting regenerative medicine treatments such as gene, cell, and tissue therapy are actively investigated. Here, multipotent mesenchymal stromal cells (MSCs), and particularly their secretomes, are of high interest due to their potency to recover and reactivate damaged or lost stem cell niches. However, a workflow for the development of MSC secretome-based products is not fully covered by regulatory authorities, and and this issue significantly complicates their clinical translation and has possibly been expressed in a huge number of failed clinical trials. One of the most critical issues in this regard relates to the development of potency assays. In this review, guidelines for biologicals and cell therapies are considered to be applied for the development of potency assays for the MSC secretome-based products that aim for tissue regeneration. Specific attention is paid to their possible effects on stem cell niches and to a spermatogonial stem cell niche in particular.

Case report: Metastatic urothelial cancer with an exceptional response to immunotherapy and comprehensive understanding of the tumor and the tumor microenvironment.

Although immune checkpoint inhibitors (ICIs) are increasingly used as second-line treatments for urothelial cancer (UC), only a small proportion of patients respond. Therefore, understanding the mechanisms of response to ICIs is critical to improve clinical outcomes for UC patients. The tumor microenvironment (TME) is recognized as a key player in tumor progression and the response to certain anti-cancer treatments. This study aims to investigate the mechanism of response using integrated genomic and transcriptomic profiling of a UC patient who was part of the KEYNOTE-045 trial and showed an exceptional response to pembrolizumab. Diagnosed in 2014 and receiving first-line chemotherapy without success, the patient took part in the KEYNOTE-045 trial for 2 years. She showed dramatic improvement and has now been free of disease for over 6 years. Recently described by Bagaev et al., the Molecular Functional (MF) Portrait was utilized to dissect genomic and transcriptomic features of the patient's tumor and TME. The patient's tumor was characterized as Immune Desert, which is suggestive of a non-inflamed microenvironment. Integrated whole-exome sequencing (WES) and RNA sequencing (RNA-seq) analysis identified an ATM mutation and high TMB level (33.9 mut/mb), which are both positive biomarkers for ICI response. Analysis further revealed the presence of the APOBEC complex, indicating the potential for use of APOBEC signatures as predictive biomarkers for immunotherapy response. Overall, comprehensive characterization of the patient's tumor and TME with the MF Portrait revealed important insights that could potentially be hypothesis generating to identify clinically useful biomarkers and improve treatment for UC patients.

Novel Potency Assay for MSC Secretome-Based Treatment of Idiopathic Male Infertility Employed Leydig Cells and Revealed Vascular Endothelial Growth Factor as a Promising Potency Marker.

Idiopathic male infertility is a highly prevalent diagnosis in developed countries with no specific treatment options. Although empirical medical treatment is widely used to restore male fertility, its efficacy remains limited and inconclusively proven. Therefore, the development of novel therapeutic approaches in this field is a high-priority task. Since the failure of testicular microenvironment components might be involved in the pathogenesis of idiopathic male infertility, application of mesenchymal stromal cells (MSCs) as well as the MSC secretome is worth considering. Previously, we showed that the intratesticular injection of MSCs or the MSC secretome led to the recovery of spermatogenesis at least through replenishing the testicular microenvironment and its maintenance by MSC-secreted paracrine factors. However, the clinical use of such products has been limited to single trials to date. This may be due to the lack of relevant potency tests reflecting mechanisms of action of the MSC secretome in male infertility models. Based on the presumptive MSC secretome mode of action on the testicular microenvironment, we suggest a novel approach to test the potential efficacy of the MSC secretome for idiopathic male infertility treatment. It represents a potency assay based on evaluation of testosterone production by isolated Leydig cells. We demonstrated that the MSC secretome stimulated testosterone secretion by Leydig cells in vitro. We then hypothesized that among the major factors of the MSC secretome, vascular endothelial growth factor (VEGF) could be responsible for the observed effects, which we confirmed by the revealed correlation between the extent of stimulated testosterone production and VEGF concentration in the MSC secretome. The pilot results obtained from the doxorubicin-induced male infertility murine model also indicate the important impact of VEGF in the MSC secretome's regenerative effects. Utilizing VEGF as a surrogate factor, a novel approach to study the potency of MSC secretome-based products for idiopathic male infertility treatment is suggested. Further validation is required for its implementation into the biopharmaceutical manufacturing process.

Regenerative medicine for male infertility: A focus on stem cell niche injury models.

Stem and progenitor cells located within stem cell niches maintain the renewal and regeneration of tissues and organs throughout the life of an adult organism. Stem cell niche component dysfunction might alter the activity of stem cells and ultimately lead to the development of difficult-to-treat chronic or acute disorders. Of note, some cases of idiopathic male infertility, a highly prevalent diagnosis with no specific treatment options, might be associated with a spermatogonial stem cell(SSC) niche disturbance. To overcome this disease entity, approaches aiming at launching the regeneration of an altered stem cell niche are worth considering. Particularly, mesenchymal stromal cells (MSCs) or their secretome might fulfill this task due to their promising contribution in recovering injured stem cell niches. However, the successful application of MSC-based treatment is limited by the uncovered mechanisms of action of MSCs and their secretome. Specific animal models should be developed or adapted to reveal the role of MSCs and their secretome in a stem cell niche recovery. In this review, in a bid to consider MSCs and their secretome as a therapeutic regenerative approach for idiopathic male infertility we focus on the rationale of SSC niche injury modeling.

MSC Secretome as a Promising Tool for Neuroprotection and Neuroregeneration in a Model of Intracerebral Hemorrhage.

Multipotent mesenchymal stromal cells (MSCs) are considered to be critical contributors to injured tissue repair and regeneration, and MSC-based therapeutic approaches have been applied to many peripheral and central neurologic disorders. It has been demonstrated that the beneficial effects of MSC are mainly mediated by the components of their secretome. In the current study, we have explored the neuroprotective potential of the MSC secretome in a rat model of intracerebral hemorrhage and shown that a 10-fold concentrated secretome of human MSC and its combination with the brain-derived neurotrophic factor (BDNF) provided a better survival and neurological outcome of rats within 14 days of intracerebral hemorrhage compared to the negative (non-treated) and positive (BDNF) control groups. We found that it was due to the ability of MSC secretome to stimulate neuron survival under conditions of glutamate-induced neurotoxicity. However, the lesion volume did not shrink in these rats, and this also correlated with prominent microglia activation. We hypothesize that this could be caused by the species-specificity of the used MSC secretome and provide evidence to confirm this. Thus, we have found that allogenic rat MSC secretome was more effective than xenogenic human MSC secretome in the rat intracerebral hemorrhage model: it reduced the volume of the lesion and promoted excellent survival and neurological outcome of the treated rats.

Total Blood Exosomes in Breast Cancer: Potential Role in Crucial Steps of Tumorigenesis.

Exosomes are crucial players in cell-to-cell communication and are involved in tumorigenesis. There are two fractions of blood circulating exosomes: free and cell-surface-associated. Here, we compared the effect of total blood exosomes (contain plasma exosomes and blood cell-surface-associated exosomes) and plasma exosomes from breast cancer patients (BCPs, n = 43) and healthy females (HFs, n = 35) on crucial steps of tumor progression. Exosomes were isolated by ultrafiltration, followed by ultracentrifugation, and characterized by cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis, and flow cytometry. Cryo-EM revealed a wider spectrum of exosome morphology with lipid bilayers and vesicular internal structures in the HF total blood in comparison with plasma. No differences in the morphology of both exosomes fractions were detected in BCP blood. The plasma exosomes and total blood exosomes of BCPs had different expression levels of tumor-associated miR-92a and miR-25-3p, induced angiogenesis and epithelial-to-mesenchymal transition (EMT), and increased the number of migrating pseudo-normal breast cells and the total migration path length of cancer cells. The multidirectional effects of HF total blood exosomes on tumor dissemination were revealed; they suppress the angiogenesis and total migration path length of MCF10A, but stimulate EMT and increase the number of migrating MCF10A and the total path length of SKBR3 cells. In addition, HF plasma exosomes enhance the metastasis-promoting properties of SKBR3 cells and stimulate angiogenesis. Both cell-free and blood cell-surface-associated exosomes are involved in the crucial stages of carcinogenesis: the initiation of EMT and the stimulation of proliferation, cell migration, and angiogenesis. Thus, for the estimation of the diagnostic/prognostic significance of circulating exosomes in the blood of cancer patients more correctly, the total blood exosomes, which consist of plasma exosomes and blood cell-surface-associated exosomes should be used.

Mesenchymal Stromal Cells as Critical Contributors to Tissue Regeneration.

Adult stem cells that are tightly regulated by the specific microenvironment, or the stem cell niche, function to maintain tissue homeostasis and regeneration after damage. This demands the existence of specific niche components that can preserve the stem cell pool in injured tissues and restore the microenvironment for their subsequent appropriate functioning. This role may belong to mesenchymal stromal cells (MSCs) due to their resistance to damage signals and potency to be specifically activated in response to tissue injury and promote regeneration by different mechanisms. Increased amount of data indicate that activated MSCs are able to produce factors such as extracellular matrix components, growth factors, extracellular vesicles and organelles, which transiently substitute the regulatory signals from missing niche cells and restrict the injury-induced responses of them. MSCs may recruit functional cells into a niche or differentiate into missing cell components to endow a niche with ability to regulate stem cell fates. They may also promote the dedifferentiation of committed cells to re-establish a pool of functional stem cells after injury. Accumulated evidence indicates the therapeutic promise of MSCs for stimulating tissue regeneration, but the benefits of administered MSCs demonstrated in many injury models are less than expected in clinical studies. This emphasizes the importance of considering the mechanisms of endogenous MSC functioning for the development of effective approaches to their pharmacological activation or mimicking their effects. To achieve this goal, we integrate the current ideas on the contribution of MSCs in restoring the stem cell niches after damage and thereby tissue regeneration.

Secretome of Mesenchymal Stromal Cells Prevents Myofibroblasts Differentiation by Transferring Fibrosis-Associated microRNAs within Extracellular Vesicles.

Fibroblasts differentiation into myofibroblasts is a central event of tissue fibrosis. Multipotent mesenchymal stromal cells (MSCs) secretome can interfere with fibrosis development; despite precise underlying mechanisms remain unclear. In this study, we tested the hypothesis that MSC secretome can affect fibroblast' differentiation into myofibroblasts by delivering regulatory RNAs, including microRNAs to these cells. Using the model of transforming growth factor-beta (TGFbeta)-induced fibroblast differentiation into myofibroblasts, we tested the activity of human MSC secretome components, specifically extracellular vesicles (MSC-EV). We showed that MSC-EV down-regulated secretion of extracellular matrix proteins by fibroblasts as well as suppressed their contractility resulting in prevention as well as reversion of fibroblasts differentiation to myofibroblasts. High-throughput sequencing of RNAs extracted from MSC-EV has revealed many fibrosis-associated microRNAs. Fibroblast treatment with MSC-EV led to direct transfer of microRNAs, which resulted in the elevation of most prominent fibrosis-associated microRNAs, including microRNA-21 and microRNA-29c. Using MSC-EV transfection by antagomirs to these microRNAs we demonstrated their involvement in the suppression of fibroblast differentiation in our model. Taken together, MSC secretome can suppress fibrosis by prevention of fibroblast differentiation into myofibroblasts as well as induce de-differentiation of the latter by direct transfer of specific microRNAs.

A magic kick for regeneration: role of mesenchymal stromal cell secretome in spermatogonial stem cell niche recovery.

BACKGROUND: Injury of stem cell niches may disturb tissue homeostasis and regeneration coordinated by specific niche components. Yet, the mechanisms of stem cell niche restoration remain poorly understood. Herein, we examined the role of mesenchymal stromal cells (MSCs) as pivotal regulators of stem cell niche recovery focusing on the effects of their secretome. METHODS: The spermatogonial stem cell (SSC) niche was selected as a model. SSC niches were injured by inducing abdominal cryptorchidism in rats. Briefly, testes of anesthetized rats were elevated into the abdominal cavity through the inguinal canal for 14 days. After descent of testes, MSC or MSC secretome treatment was applied to the animals by local subtunical injections. RESULTS: Local administration of MSC or MSC secretome was sufficient to recover spermatogenesis and production of functional germ cells. The effects of MSC and their secreted components were comparable, leading to restoration of Sertoli cell pools and recovery of Leydig cell secretory functions. CONCLUSION: Our data suggest that MSCs mimic the functions of lost supportive cells within the stem cell niche, transiently providing paracrine stimuli for target cells and triggering tissue regenerative processes after damage.

Conditioned Medium from Human Mesenchymal Stromal Cells: Towards the Clinical Translation.

Mesenchymal stem/stromal cells (MSC) remain a promising tool for regenerative medicine as the efficacy of MSC-based cell therapy has been demonstrated for a broad spectrum of indications. Their therapeutic potency is mainly associated with their ability to secrete multiple factors critical for tissue regeneration. Due to comparable effects along with superior safety MSC conditioned medium (MSC-CM) containing a complex of MSC-secreted products is considered a reasonable alternative to cell therapy. However, the lack of standards regulating bioprocessing, use of proper auxiliary materials, and quality control complicates the development of MSC secretome-based therapeutics. In this study, we suggested several approaches addressing these issues. We manufactured 36 MSC-CM samples based on different xeno-free serum-free chemically defined media (DMEM-LG or MSC NutriStem® XF) using original protocols and considered total concentrations of regeneration-associated paracrine factors secreted by human adipose-derived MSC at each time-point of conditioning. Using regression analysis, we retrospectively predicted associations between concentrations of several components of MSC-CM and its biological activity to stimulate human dermal fibroblast and endothelial cell migration in vitro as routine examples of potency assays for cell-based products. We also demonstrated that the cell culture medium might affect MSC-CM biological activity to varying degrees depending on the potency assay type. Furthermore, we showed that regression analysis might help to overcome donor variability. The suggested approaches might be successfully applied for other cell types if their secretome was shown to be promising for application in regenerative medicine.