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Mesenchymal stromal cells (MSC) represent a promising treatment option for tendon disorders and joint diseases, primarily osteoarthritis. Since MSC are highly context-sensitive to their microenvironment, their therapeutic efficacy is influenced by their tissue-specific pathologically altered targets. These include not only cellular components, such as resident cells and invading immunocompetent cells, but also components of the tissue-characteristic extracellular matrix. Although numerous in vitro models have already shown potential MSC-related mechanisms of action in tendon and joint diseases, only a limited number reflect the disease-specific microenvironment and allow conclusions about well-directed MSC-based therapies for injured tendon and joint-associated tissues. In both injured tissue types, inflammatory processes play a pivotal pathophysiological role. In this context, MSC-mediated macrophage modulation seems to be an important mode of action across these tissues. Additional target cells of MSC applied in tendon and joint disorders include tenocytes, synoviocytes as well as other invading and resident immune cells. It remains of critical importance whether the context-sensitive interplay between MSC and tissue- and disease-specific targets results in an overall promotion or inhibition of the desired therapeutic effects. This review presents the authors’ viewpoint on disease-related targets of MSC therapeutically applied in tendon and joint diseases, focusing on the equine patient as valid animal model.

Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine adipose-derived (AD-) MSC culture so far, as it supported AD-MSC proliferation and basic characteristics. The aim of the current study was to further analyze the functional properties of equine AD-MSC cultured with the same ePL, focusing on cell fitness, genetic stability and pro-angiogenic potency. All experiments were performed with AD-MSC from n = 5 horses, which were cultured either in medium supplemented with 10% FBS, 10% ePL or 2.5% ePL. AD-MSC cultured with 2.5% ePL, which previously showed decreased proliferation potential, displayed higher apoptosis but lower senescence levels as compared to 10% ePL medium (p < 0.05). Non-clonal chromosomal aberrations occurred in 8% of equine AD-MSC cultivated with FBS and only in 4.8% of equine AD-MSC cultivated with 10% ePL. Clonal aberrations in the AD-MSC were neither observed in FBS nor in 10% ePL medium. Analysis of AD-MSC and endothelial cells in an indirect co-culture revealed that the ePL supported the pro-angiogenic effects of AD-MSC. In the 10% ePL group, more vascular endothelial growth factor (VEGF-A) was released and highest VEGF-A concentrations were reached in the presence of ePL and co-cultured cells (p < 0.05). Correspondingly, AD-MSC expressed the VEGF receptor-2 at higher levels in the presence of ePL (p < 0.05). Finally, AD-MSC and 10% ePL together promoted the growth of endothelial cells and induced the formation of vessel-like structures in two of the samples. These data further substantiate that buffy-coat-based ePL is a valuable supplement for equine AD-MSC culture media. The ePL does not only support stable equine AD-MSC characteristics as demonstrated before, but it also enhances their functional properties.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellularmatrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of Nacetyl- galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

Simple Summary Regenerative medicine using platelet-based blood products or adult stem cells offers the prospect of better clinical outcomes with many diseases. In veterinary medicine, most progress has been made with the development and therapeutic use of these regenerative therapeutics in horses, but the clinical need is given in dogs as well. Our aim was to transfer previous advances in the development of horse regenerative therapeutics, specifically the use of platelet lysate for feeding stem cell cultures, to the dog. Here, we describe the scalable production of canine platelet lysate, which could be used in regenerative biological therapies. We also evaluated the canine platelet lysate for its suitability in feeding canine stem cell cultures in comparison to equine platelet lysate used for equine stem cell cultures. Platelet lysate production from canine blood was successful, but the platelet lysate did not support stem cell culture in dogs in the same beneficial way observed with the equine platelet lysate and stem cells. In conclusion, canine platelet lysate can be produced in large scales as described here, but further research is needed to improve the cultivation of canine stem cells. Abstract Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

Mesenchymal stromal cell (MSC) transplantation ameliorated hepatic lipid load; tissue inflammation; and fibrosis in rodent animal models of non-alcoholic steatohepatitis (NASH) by as yet largely unknown mechanism(s). In a mouse model of NASH; we transplanted bone marrow-derived MSCs into the livers; which were analyzed one week thereafter. Combined metabolomic and proteomic data were applied to weighted gene correlation network analysis (WGCNA) and subsequent identification of key drivers. Livers were analyzed histologically and biochemically. The mechanisms of MSC action on hepatocyte lipid accumulation were studied in co-cultures of hepatocytes and MSCs by quantitative image analysis and immunocytochemistry. WGCNA and key driver analysis revealed that NASH caused the impairment of central carbon; amino acid; and lipid metabolism associated with mitochondrial and peroxisomal dysfunction; which was reversed by MSC treatment. MSC improved hepatic lipid metabolism and tissue homeostasis. In co-cultures of hepatocytes and MSCs; the decrease of lipid load was associated with the transfer of mitochondria from the MSCs to the hepatocytes via tunneling nanotubes (TNTs). Hence; MSCs may ameliorate lipid load and tissue perturbance by the donation of mitochondria to the hepatocytes. Thereby; they may provide oxidative capacity for lipid breakdown and thus promote recovery from NASH-induced metabolic impairment and tissue injury.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease and one of the most severe complications that develop in premature infants following mechanical ventilation, exposure to supplemental oxygen, and inflammation. The hallmarks of the lung pathology are arrested lung development, including fewer and larger alveoli with less septation, thickening of alveolar septa, and impaired development of the capillary network. BPD is associated with increased mortality, respiratory morbidity, neurodevelopmental impairment, and increased healthcare costs. Significant advancements in neonatology in the last several decades, including antenatal steroids and exogenous surfactant replacement therapy, more gentle ventilation methods, and judicious oxygen use, have allowed for the survival of more preterm infants. However, the incidence of BPD still remains high and currently, there is no cure for the disease. Novel effective interventions at this stage of life are of exceptional value. Considering their great potential in promoting tissue regeneration and modulating inflammation, mesenchymal stromal cells (MSCs) represent a promising avenue for treating several disorders, including BPD. Umbilical cord-derived MSCs (UC-MSCs) offer biological advantages over other MSC sources (easily available, high proliferative capacity, and better repair efficacy). Pioneering work in our lab showed that MSCs prevent injury to the developing lung in a rat model mimicking BPD. However, there are still considerable challenges that must be overcome before MSCs can be effectively implemented in clinical trials. As such, UC-MSC heterogeneity is poorly understood, with concerns regarding variations from donors and batches. Thus, to improve the reproducibility of basic research and clinical applications, and to identify the optimal therapeutic cell product, better molecular characterization of UC-MSCs and the development of standardized BPD models will be essential in the clinical translation of MSC therapy for BPD. Moreover, considering that BPD is a disease of prematurity, the therapeutic potential of UC-MSCs isolated from preterm birth is of major interest. In the study presented here, using single-cell RNA sequencing (scRNA-seq), we characterized MSCs isolated from the UC of term and preterm pregnancies at delivery (term and preterm donors), as well as non-progenitor control cell line, human neonatal dermal fibroblasts (HNDFs). Moreover, we associated UC-MSC transcriptomic profiles with their therapeutic potential in hyperoxia-induced lung injury in neonatal rats. Finally, we developed and characterized a novel two-hit (2HIT) BPD model in neonatal mice, assessed UC-MSCs' optimal route of injection, timing, and dose, and evaluated their therapeutic effects in that model. We showed that UC-MSCs isolated from the majority of term and preterm donors, including preterm donors with pregnancy-related complications, have limited heterogeneity and possessed a transcriptome enriched in genes related to cell cycle and cell proliferation activity (termed "progenitor-like" cells). In contrast, UC-MSCs isolated from one term and two preterm donors with preeclampsia displayed a unique transcriptome comprised of many genes related to fibroblast activity, including extracellular matrix (ECM) organization (termed "fibroblast-like" cells). In addition, treatment with progenitor-like UC-MSCs, but not with fibroblast-like cells nor HNDFs, significantly improved lung structure, function, and pulmonary hypertension (PH) in hyperoxia-induced lung injury in neonatal rats. We identified marker genes for the therapeutic UC-MSCs (progenitor-like cells) and non-therapeutic cells (fibroblast-like cells and HNDFs). Among them, the high expression of major histocompatibility complex class I (MHCI) is associated with a reduced therapeutic effect. Furthermore, we developed a novel 2HIT BPD mice model with in-depth characterization of the innate immune response and lung injury. 2HIT injury caused a transient type 1 proinflammatory cytokine response and a significant decrease in type 2 anti-inflammatory cytokine lung expression and number of anti-inflammatory M2 type alveolar macrophages. Moreover, 2HIT mice showed impaired lung compliance and growth. Repeated intravenous (i.v.) injections of UC-MSCs at a dose of 20×10⁶ cells/kg body weight (BW) on postnatal day (PD) one and two improved survival, BW, lung compliance, and growth of 2HIT animals. In conclusion, scRNA-seq experimentation provided evidence that UC-MSCs isolated from different donors harbor different transcriptomes with progenitor-like or fibroblast-like characteristics. Only progenitor-like cells provided a therapeutic effect in hyperoxia-induced lung injury in neonatal rats. The development of a novel murine 2HIT BPD model allowed us to characterize the innate immune response and lung pathology and confirm the optimal dose of UCMSCs to provide therapeutic potential in that model. These results will enable better therapeutic selection of UC-MSCs and help improve treatment regimen prior to ultimate clinical translation.

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor b1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/ methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LCMS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.

Radiotherapy has major therapeutic benefits for cancer patients, but ionizing radiation causes damage of surrounding healthy tissues with poor wound healing a common side effect. Therefore, further oncoplastic, reconstructive surgery is challenging and often problematic. Current research models use normal human dermal fibroblasts irradiated in vitro to mimic radiation damage, but this is not comparable to ionising radiation and only measures acute changes. Since radiotherapy may induce epigenetic changes leading to alterations in dermal fibroblast phenotype, the first aim of this study was to compare fibroblasts cultured from irradiated skin with non-irradiated skin. As mesenchymal stem cells isolated from adipose tissue may offer beneficial effects in the regenerative capacity of irradiated tissue, the second part of this study was to compare those cultured from non-irradiated and irradiated breast tissue. Histological changes in the structural organisation of breast tissue in situ from donors exposed to radiotherapy was compared to untreated breast. Primary cultures of dermal fibroblasts from irradiated and non-irradiated breast skin were established and comparisons quantitated in proliferation (CyQuant), metabolism (Alamar Blue), migration (scratch-wound assay), collagen production (Sircol), levels of proteases and protease inhibitors (human protease/protease inhibitor array) and gene expression of COL1A1, COL3A1, MMP1, MMP2, TIMP1 and PPAR-γ mRNA (qPCR). Cells from the stromal vascular fraction (SVF) were cultured and characterised by immunocytochemistry and compared to human preadipocytes sourced commercially. The secretion of FGF, adiponectin and VEGF by the preadipocyte and the SVF mesenchymal cells was compared and the ability of their secretome to modulate dermal fibroblast proliferation, metabolism and migration was evaluated. Radiotherapy caused extensive disorganisation of the reticular dermis and flattening of the epidermal-dermal junction. Dermal fibroblasts cultured from irradiated skin had a pronounced spindle shaped morphology with longer thinner projections and took approximately twice as long to explant and grow. They had a lower proliferative and higher basal metabolic rate and did not respond to FGF-2. While they secreted similar amounts of total collagen they demonstrated distinct differences in proteolytic enzyme and protease inhibitor expression. This is the first report to culture cells from the SVF of irradiated breast tissue. The cells expressed the preadipocyte markers CD10, CD73 and CD105 and no CD45 (negative marker). SVF cells cultured displayed a typical ASC fibroblastoid morphology. Analysis of the secretome identified the presence of FGF, adiponectin and VEGF, while functional analysis demonstrated a stimulatory effect on normal dermal fibroblast migration, although irradiated dermal fibroblasts were unresponsive. Radiotherapy induces long term, detrimental changes in breast skin. This is the first quantitative characterisation of dermal fibroblasts and mesenchymal cells from the SVF, subjected to ionising radiation in situ. Changes in their phenotype that alter their function will impact on wound healing. Further characterisation of these cells may explain their dysfunctional behaviour, and lead to therapies to reverse or reduce this deleterious side-effect and significantly improve treatments facilitating wound healing following radiation injury. / Plastic Surgery and Burns Research unit