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Following a one medicine approach, the development of regenerative therapies for human patients leads to innovative treatments for animals, while pre-clinical studies on animals provide knowledge to advance human medicine. Among many different biological products under investigation, stem cells are among the most prominent. Mesenchymal stromal cells (MSCs) are extensively investigated, but they present challenges such as senescence and limited differentiation ability. Embryonic stem cells (ESCs) are pluripotent cells with a virtually unlimited capacity for self-renewal and differentiation, but the use of embryos carries ethical concerns. Induced pluripotent stem cells (iPSCs) can overcome all of these limitations, as they closely resemble ESCs but are derived from adult cells by reprogramming in the laboratory using pluripotency-associated transcription factors. iPSCs hold great potential for applications in therapy, disease modeling, drug screening, and even species preservation strategies. However, iPSC technology is less developed in veterinary species compared to human. This review attempts to address the specific challenges associated with generating and applying iPSCs from companion animals. Firstly, we discuss strategies for the preparation of iPSCs in veterinary species and secondly, we address the potential for different applications of iPSCs in companion animals. Our aim is to provide an overview on the state of the art of iPSCs in companion animals, focusing on equine, canine, and feline species, as well as to identify which aspects need further optimization and, where possible, to provide guidance on future advancements. Following a “step-by-step” approach, we cover the generation of iPSCs in companion animals from the selection of somatic cells and the reprogramming strategies, to the expansion and characterization of iPSCs. Subsequently, we revise the current applications of iPSCs in companion animals, identify the main hurdles, and propose future paths to move the field forward. Transferring the knowledge gained from human iPSCs can increase our understanding in the biology of pluripotent cells in animals, but it is critical to further investigate the differences among species to develop specific approaches for animal iPSCs. This is key for significantly advancing iPSC application in veterinary medicine, which at the same time will also allow gaining pre-clinical knowledge transferable to human medicine.

The recent interest in advanced biologic therapies in veterinary medicine has opened up opportunities for new treatment modalities with considerable clinical potential. Studies with mesenchymal stromal cells (MSCs) from animal species have focused on in vitro characterization (mostly following protocols developed for human application), experimental testing in controlled studies and clinical use in veterinary patients. The ability of MSCs to interact with the inflammatory environment through immunomodulatory and paracrine mechanisms makes them a good candidate for treatment of inflammatory musculoskeletal conditions in canine species. Analysis of existing data shows promising results in the treatment of canine hip dysplasia, osteoarthritis and rupture of the cranial cruciate ligament in both sport and companion animals. Despite the absence of clear regulatory frameworks for veterinary advanced therapy medicinal products, there has been an increase in the number of commercial cell-based products that are available for clinical applications, and currently the commercial use of veterinary MSC products has outpaced basic research on characterization of the cell product. In the absence of quality standards for MSCs for use in canine patients, their safety, clinical efficacy and production standards are uncertain, leading to a risk of poor product consistency. To deliver high-quality MSC products for veterinary use in the future, there are critical issues that need to be addressed. By translating standards and strategies applied in human MSC manufacturing to products for veterinary use, in a collaborative effort between stem cell scientists and veterinary researchers and surgeons, we hope to facilitate the development of quality standards. We point out critical issues that need to be addressed, including a much higher level of attention to cell characterization, manufacturing standards and release criteria. We provide a set of recommendations that will contribute to the standardization of cell manufacturing methods and better quality assurance.

In recent years mesenchymal stromal cells (MSCs) have received a great deal of interest for the treatment of major diseases, but clinical translation and market authorization have been slow. This has been due in part to a lack of standardization in cell manufacturing protocols, as well as a lack of biologically meaningful cell characterization tools and release assays. Cell production strategies to date have involved complex manual processing in an open environment which is costly, inefficient and poses risks of contamination. The NANT 001 bioreactor has been developed for the automated production of small to medium cell batches for autologous use. This is a closed, benchtop system which automatically performs several processes including cell seeding, media change, real-time monitoring of temperature, pH, cell confluence and cell detachment. Here we describe a validation of the bioreactor in an environment compliant with current good manufacturing practice (cGMP) to confirm its utility in replacing standardized manual processing. Stromal vascular fraction (SVF) was isolated from lipoaspirate material obtained from healthy donors. SVF cells were seeded in the bioreactor. Cell processing was performed automatically and cell harvesting was triggered by computerized analysis of images captured by a travelling microscope positioned beneath the cell culture flask. For comparison, the same protocol was performed in parallel using manual methods. Critical quality attributes (CQA) assessed for cells from each process included cell yield, viability, surface immunophenotype, differentiation propensity, microbial sterility and endotoxin contamination. Cell yields from the bioreactor cultures were comparable in the manual and automated cultures and viability was >90% for both. Expression of surface markers were consistent with standards for adipose-derived stromal cell (ASC) phenotype. ASCs expanded in both automated and manual processes were capable of adipogenic and osteogenic differentiation. Supernatants from all cultures tested negative for microbial and endotoxin contamination. Analysis of labor commitment indicated considerable economic advantage in the automated system in terms of operator, quality control, product release and management personnel. These data demonstrate that the NANT 001 bioreactor represents an effective option for small to medium scale, automated, closed expansion of ASCs from SVF and produces cell products with CQA equivalent to manual processes.

ObjectivesMesenchymal stem/stromal cells (MSCs) are widely investigated in regenerative medicine thanks to their immunomodulatory properties. They exert their anti-inflammatory function thanks to the secretion of a number of mediators, including proteins and miRNAs, which can be released in the extracellular environment or in the cargo of extracellular vesicles (EVs). However, the role of miRNAs in the suppressive function of MSCs is controversial. The aim of the study was to identify miRNAs that contribute to the immunomodulatory function of human bone marrow-derived MSCs (BM-MSCs).MethodsHuman BM-MSCs were primed by coculture with activated peripheral blood mononuclear cells (aPBMCs). High throughput miRNA transcriptomic analysis was performed using Human MicroRNA TaqMan® Array Cards. The immunosuppressive function of miRNAs was investigated in mixed lymphocyte reactions and the delayed type hypersensitivity (DTH) murine model.ResultsUpon priming, 21 out of 377 tested miRNAs were significantly modulated in primed MSCs. We validated the up-regulation of miR-29a, miR-146a, miR-155 and the down-regulation of miR-149, miR-221 and miR-361 in additional samples of primed MSCs. We showed that miR-155 significantly reduced the proliferation of aPBMCs in vitro and inflammation in vivo, using the DTH model. Analysis of miRNA-mRNA interactions revealed miR-221 as a potential target gene that is down-regulated by miR-155 both in primed MSCs and in aPBMCs.ConclusionHere, we present evidence that miR-155 participates to the immunosuppressive function of human BM-MSCs and down-regulates the expression of miR-221 as a possible inflammatory mediator.

A fundamental structural component of extracellular matrix in all connective and interstitial tissue, collagen is the most abundant protein in the human body. To date, mammalian collagens sources represent the golden standard for multiple biomedical applications, while marine-derived collagens have largely been used in industry (food, pharmaceutical, and cosmetic), with little use in research and clinical applications. Herein we demonstrate the effective use Rhizostoma pulmo jellyfish collagen, a source of biocompatible, sustainable collagen for 2D and 3D cell culture, addressing the global drive for technological developments that result in the replacement of animals and their derived products in research. Jellyfish collagen harbors similar structural features mammalian collagen type I, despite differing slightly in amino acid content. Jellyfish collagen supports ovarian cancer (OvCa) cell line proliferation, cellular morphology and expression of epithelial to mesenchymal transition markers, supporting the use of R. pulmo as a non-mammalian collagen cell culture substrate. Furthermore, R. pulmo collagen is effective in 3D device fabrication such as sponges where it mimics tissue architecture complexity. OvCa cells migrated and differentiated within the R. pulmo collagen 3D scaffolds confirming its suitability for advanced cell culturing applications, providing an excellent alternative to mammalian collagen sources for the culture of human cells.

Osteoarthritis (OA) is an inflammatory condition still lacking effective treatments. Mesenchymal stem/stromal cells (MSCs) have been successfully employed in pre-clinical models aiming to resurface the degenerated cartilage. In early-phase clinical trials, intra-articular (IA) administration of MSCs leads to pain reduction and cartilage protection or healing. However, the consistent lack of engraftment indicates that the observed effect is delivered through a “hit-and-run” mechanism, by a temporal release of paracrine molecules. MSCs express a variety of chemokines and cytokines that aid in repair of degraded tissue, restoration of normal tissue metabolism and, most importantly, counteracting inflammation. Secretion of therapeutic factors is increased upon licensing by inflammatory signals or apoptosis, induced by the host immune system. Trophic effectors are released as soluble molecules or carried by extracellular vesicles (ECVs). This review provides an overview of the functions and mechanisms of MSC-secreted molecules found to be upregulated in models of OA, whether using in vitro or in vivo models.