Investigating the paracrine and juxtacrine abilities of adipose-derived stromal cells in angiogenesis triple cell co-cultures.

The pro-angiogenic abilities of adipose-derived stromal cells (ASCs) make them attractive candidates for cellular therapy, especially for ischemic disease indications. However, details regarding the underlying mechanisms remain elusive. Therefore, this study aimed to investigate paracrine and juxtacrine abilities of ASCs in angiogenesis triple cell co-cultures by detailed image analysis of the vascular-like structures. Fibroblast-endothelial cell co-cultures were established, and ASCs were added directly or indirectly through inserts. The cultures were treated with antibodies or subjected to analyses using ELISA and RT2 PCR Arrays. The model consistently generated vascular-like structures. ASCs increased the total branch lengths equally well in paracrine and juxtacrine conditions, by increasing the number of branches and average branch lengths (ABL). In contrast, addition of VEGF to the model increased the number of branches, but not the ABL. Still, ASCs increased the VEGF levels in supernatants of paracrine and juxtacrine co-cultures, and anti-VEGF treatment decreased the sprouting. ASCs themselves up-regulated collagen type V in response to paracrine signals from the co-cultures. The results suggest that ASCs initiate sprouting through secretion of several paracrine factors, among which VEGF is identified, but VEGF alone does not recapitulate the paracrine actions of ASCs. By employing neutralizing antibodies and dismantling common model outputs using image analysis, the triple cell co-culture is an attractive tool for discovery of the paracrine factors in ASCs' secretome which act in concert with VEGF to improve angiogenesis.

Mesenchymal Stromal/Stem Cell Therapy Improves Salivary Flow Rate in Radiation-Induced Salivary Gland Hypofunction in Preclinical in vivo Models: A Systematic Review and Meta-Analysis.

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) have been suggested for salivary gland (SG) restoration following radio-induced salivary gland damage. This study aimed to determine the safety and effectiveness of MSC therapy on radio-induced SG damage and hypofunction in preclinical in vivo studies. METHODS: PubMed and EMBASE were systematically searched for preclinical in vivo interventional studies evaluating efficacy and safety of MSC treatment following radio-induced salivary gland damage published before 10th of January 2022. The primary endpoint was salivary flow rate (SFR) evaluated in a meta-analysis. The study protocol was published and registered on PROSPERO ( www.crd.ac.uk/prospero ), registration number CRD42021227336. RESULTS: A total of 16 preclinical in vivo studies were included for qualitative analysis (858 experimental animals) and 13 in the meta-analysis (404 experimental animals). MSCs originated from bone marrow (four studies), adipose tissue (10 studies) and salivary gland tissue (two studies) and were administered intravenously (three studies), intra-glandularly (11 studies) or subcutaneously (one study). No serious adverse events were reported. The overall effect on SFR was significantly increased with a standardized mean difference (SMD) of 6.99 (95% CI: 2.55-11.42). Studies reported improvements in acinar tissue, vascular areas and paracrine factors. CONCLUSION: In conclusion, this systematic review and meta-analysis showed a significant effect of MSC therapy for restoring SG functioning and regenerating SG tissue following radiotherapy in preclinical in vivo studies without serious adverse events. MSC therapy holds significant therapeutic potential in the treatment of radio-induced xerostomia, but comprehensive, randomized, clinical trials in humans are required to ascertain their efficacy in a clinical setting.

Adipose-derived stromal cells increase the formation of collagens through paracrine and juxtacrine mechanisms in a fibroblast co-culture model utilizing macromolecular crowding.

BACKGROUND: Adipose-derived stromal cells (ASCs) possess a multitude of regenerative capabilities, which include immunomodulation, angiogenesis, and stimulation of extracellular matrix (ECM) remodeling. However, the underlying mechanisms leading to ECM remodeling remain largely elusive and highlight the need for functional in vitro models for mode of action studies. Therefore, the purpose of this study was to develop an in vitro co-culture model to investigate the capabilities of ASCs to modulate fibroblasts and ECM. METHODS: An ECM in vitro model with ASCs and normal human dermal fibroblasts (NHDFs) was established utilizing macromolecular crowding, ascorbic acid, and TGF-ß stimulation. Paracrine and juxtacrine co-cultures were created using transwell inserts and cell cultures with direct cell-cell contacts. The cultures were screened using RT2 PCR Profiler Arrays; the protein levels of myofibroblast differentiation marker alpha smooth muscle actin (αSMA) and ECM remodeling enzymes were analyzed using western blot on cell lysates; the formation of collagen type I, III, VI, and fibronectin was investigated using ELISA on culture supernatants; and the deposition of collagens was analyzed using immunocytochemistry. RESULTS: TGF-ß stimulation of NHDF monocultures increased the expression of 18 transcripts relevant for ECM formation and remodeling, the protein levels of αSMA and matrix metalloproteinase-2 (MMP-2), the formation of collagen type I, III, VI, and fibronectin, and the deposition of collagen type I and VI and decreased the protein levels of MMP-14. Inclusion of ASCs in the ECM co-culture model increased the formation of collagen type I and III through paracrine mechanisms and the formation of collagen type VI through juxtacrine mechanisms. CONCLUSIONS: The co-culture model provides effective stimulation of NHDF monocultures by TGF-ß for enhanced formation and deposition of ECM. In the model, ASCs induce changes in ECM by increasing formation of collagen type I, III and VI. The obtained results could guide further investigations of ASCs' capabilities and underlying mechanisms related to ECM formation and remodeling.

Mesenchymal stromal/stem cell therapy for radiation-induced salivary gland hypofunction in animal models: a protocol for a systematic review and meta-analysis.

BACKGROUND: Salivary gland (SG) hypofunction (objectively reduced saliva flow rate) and xerostomia (subjective sensation of dry mouth) are common and burdensome side effects of radiotherapy to the head and neck region. Currently, only sparse symptomatic treatment is available to ease the discomfort of xerostomia. The objective of this study is to assess the effect of mesenchymal stem cell (MSC) therapy on SG function after radiation-induced injury. METHODS: This systematic review will include animal intervention studies assessing efficacy and safety of MSCs in treating radiation-induced SG hypofunction. The primary outcome is the effect of MSC administration on salivary flow rates (SFR), by comparing treated groups to control groups when available. Secondary outcomes are morphological and immunohistochemical effects as well as safety of MSC treatment. Electronic searches in MEDLINE (PubMed) and Embase databases will be constructed and validated according to the peer review of electronic search strategies (PRESS) and assessed by two independent researchers. Data from eligible studies will be extracted, pooled, and analyzed using random-effects models. Risk of bias will be evaluated with the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool. DISCUSSION: Thus far, critical appraisal of MSC therapy as an effective treatment for SG hypofunction caused solely by radiation injury has not been conducted. A summary of the existing literature on preclinical studies concerning this issue can provide valuable information about effectiveness, mode of action, and safety, allowing further optimization of preclinical and clinical trials. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42021227336.

The Initial Cardiac Tissue Response to Cryopreserved Allogeneic Adipose Tissue-Derived Mesenchymal Stromal Cells in Rats with Chronic Ischemic Cardiomyopathy.

Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2+ and CD38+ macrophages (p = 0.047 and p = 0.021), as well as in the CD4/CD8 lymphocyte ratio (p = 0.021), were found in the ASC group compared to the saline group. This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.