OBJECTIVE: To investigate mechanistically the reported beneficial effects of immune-activated mesenchymal stromal cell (MSC) therapy to treat equine septic arthritis, leveraging Nanostring technology.
METHODS: 8 Quarter Horses with induced tibiotarsal Staphylococcus aureus septic arthritis treated IA with either Toll-like receptor-3 agonist polyinosinic:polycytidylic acid-activated MSCs + vancomycin antimicrobials (TLR-MSC-VAN; n = 4) or antimicrobials (VAN; 4).
METHODS: Synovial tissues were collected and fixed in neutral-buffered 10% formalin, and formalin-fixed paraffin-embedded synovial and osteochondral tissues were sequenced using a custom-designed 200-gene equine Nanostring nCounter immune panel to directly quantify expression of key immune and cartilage-related genes. Immunohistochemistry to detect CD3+ T cells was performed on synovial tissues to further quantify T-cell infiltration in TLR-MSC-VAN- versus VAN-treated joints.
RESULTS: Comparison of synovial transcriptomes between groups revealed moderate changes in differential gene expression, with upregulated expression of 9 genes and downregulated expression of 17 genes with fold change ≥ 2 or ≤ -2 and a significant false discovery rate-adjusted P value of ≤ .05. The most upregulated genes in TLR-MSC-VAN-treated horses included those related to T-lymphocyte recruitment and function, while pathways related to innate immune activation and inflammation were significantly downregulated. Immunohistochemistry and quantitation of CD3+ T-cell infiltrates revealed a numerically greater infiltrate in synovial tissues of TLR-MSC-VAN-treated horses, which did not reach statistical significance in this small sample set (P = .20).
CONCLUSIONS: Targeted transcriptomic analyses using an equine Nanostring immune and cartilage health panel provided new mechanistic insights into how innate and adaptive immune cells within synovial tissues respond to TLR-activated MSC treatment when used to treat septic arthritis.

Mesenchymal stromal cell (MSC) therapy is a promising treatment that allows for drug minimization in clinical kidney transplantation. While it is thought that MSCs rapidly go into apoptosis after infusion, clinical evidence for this is scarce since methods to detect cell death of infused cells in vivo are lacking. Cell-free DNA (cfDNA) has recently gained attention as a biomarker for cell death.
In this study, we longitudinally measured cfDNA in plasma samples of the recipient, kidney donor, and allogeneic third-party MSC in the context of the Neptune study. cfDNA levels were measured at several time points before and after allogeneic MSC infusion in the 10 recipients who participated in the Neptune study. cfDNA ratios between the recipient, kidney graft, and MSC were determined.
We observed a peak in MSC-derived cfDNA 4 h after the first and second infusions, after which MSC-derived cfDNA became undetectable. Generally, kidney graft-derived cfDNA remained in the baseline-level range.
Our results support preclinical data that MSC are short-lived after infusion, also in a clinical in vivo setting, and are relevant for further research into the mechanism of action of MSC therapy.

Cell therapy using mesenchymal stromal cells (MSCs) is being studied for its immunosuppressive effects. In organ transplantation, the amount of MSCs that accumulate in transplanted organs and other organs may differ depending on administration timing, which may impact their immunosuppressive effects. In vitro, adipose-derived mesenchymal stem cells (ADMSCs) suppress lymphocyte activation under cell-to-cell contact conditions. However, in vivo, it is controversial whether ADMSCs are more effective in accumulating in transplanted organs or in secondary lymphoid organs. Herein, we aimed to investigate whether the timing of ADMSC administration affects its immunosuppression ability in a rat lung transplantation model. In the transplantation study, rats were intramuscularly administered half the usual dose of tacrolimus (0.5 mg/kg) every 24 h after lung transplantation. ADMSCs (1 × 106) were administered via the jugular vein before (PreTx) or after (PostTx) transplantation. Cell tracking using quantum dots was performed. ADMSCs accumulated predominantly in the lung and liver; fewer ADMSCs were distributed in the grafted lung in the PreTx group than in the PostTx group. The rejection rate was remarkably low in the ADMSC-administered groups, particularly in the PostTx group. Serum tumor necrosis factor-α (TNF-α), interferon-γ, and interleukin (IL)-6 levels showed a greater tendency to decrease in the PreTx group than in the PostTx group. The proportion of regulatory T cells in the grafted lung 10 days after transplantation was higher in the PostTx group than in the PreTx group. PostTx administration suppresses rejection better than PreTx administration, possibly due to regulatory T cell induction by ADMSCs accumulated in the transplanted lungs, suggesting a mechanism different from that in heart or kidney transplantation that PreTx administration is more effective than PostTx administration. These results could help establish cell therapy using MSCs in lung transplantation.

The development of graft versus host disease (GVHD) represents a long-standing complication of allogeneic hematopoietic cell transplantation (allo-HCT). Different approaches have been used to control the development of GVHD with most relying on variations of chemotherapy drugs to eliminate allo-reactive T cells. While these approaches have proven effective, it is generally accepted that safer, and less toxic GVHD prophylaxis drugs are required to reduce the health burden placed on allo-HCT recipients. In this review, we will summarize the emerging concepts revolving around three biologic-based therapies for GVHD using T regulatory cells (Tregs), myeloid-derived-suppressor-cells (MDSCs) and mesenchymal stromal cell (MSC) exosomes. This review will highlight how each specific modality is unique in its mechanism of action, but also share a common theme in their ability to preferentially activate and expand Treg populations in vivo. As these three GVHD prevention/treatment modalities continue their path toward clinical application, it is imperative the field understand both the biological advantages and disadvantages of each approach.

Pelvic floor dysfunction (PFDs), which include pelvic organ prolapse (POP), stress urinary incontinence (SUI) and anal incontinence (AI), are common degenerative diseases in women that have dramatic effects on quality of life. The pathology of PFDs is based on impaired pelvic connective tissue supportive strength due to an imbalance in extracellular matrix (ECM) metabolism, the loss of a variety of cell types, such as fibroblasts, muscle cells, peripheral nerve cells, and oxidative stress and inflammation in the pelvic environment. Fortunately, exosomes, which are one of the major secretions of mesenchymal stromal cells (MSCs), are involved in intercellular communication and the modulation of molecular activities in recipient cells via their contents, which are bioactive proteins and genetic factors such as mRNAs and miRNAs. These components modify fibroblast activation and secretion, facilitate ECM modelling, and promote cell proliferation to enhance pelvic tissue regeneration. In this review, we focus on the molecular mechanisms and future directions of exosomes derived from MSCs that are of great value in the treatment of PFD.

UNASSIGNED: Rapid development of antibiotic resistance necessitates advancement of novel therapeutic strategies to treat infection. Mesenchymal stromal cells (MSC) possess antimicrobial and immunomodulatory properties, mediated through antimicrobial peptide secretion and recruitment of innate immune cells including neutrophils and monocytes. TLR-3 activation of human, canine and equine MSC has been shown to enhance bacterial killing and clearance in vitro, in rodent Staphylococcal biofilm infection models and dogs with spontaneous multi-drug-resistant infections. The objective of this study was to determine if intra-articular (IA) TLR-3-activated MSC with antibiotics improved clinical parameters and reduced bacterial counts and inflammatory cytokine concentrations in synovial fluid (SF) of horses with induced septic arthritis.
UNASSIGNED: Eight horses were inoculated in one tarsocrural joint with multidrug-resistant Staphylococcus aureus (S. aureus). Bone marrow-derived MSC from three unrelated donors were activated with TLR-3 agonist polyinosinic, polycytidylic acid (pIC). Recipient horses received MSC plus vancomycin (TLR-MSC-VAN), or vancomycin (VAN) alone, on days 1, 4, 7 post-inoculation and systemic gentamicin. Pain scores, quantitative bacterial counts (SF, synovium), SF analyses, complete blood counts, cytokine concentrations (SF, plasma), imaging changes (MRI, ultrasound, radiographs), macroscopic joint scores and histologic changes were assessed. Results were reported as mean ± SEM.
UNASSIGNED: Pain scores (d7, P=0.01, 15.2±0.2 vs. 17.9±0.5), ultrasound (d7, P=0.03, 9.0±0.6 vs. 11.8±0.5), quantitative bacterial counts (SF d7, P=0.02, 0±0 vs. 3.4±0.4; synovium P=0.003, 0.4±0.4 vs. 162.7±18.4), systemic neutrophil (d4, P=0.03, 4.6±0.6 vs. 7.8±0.6) and serum amyloid A (SAA) (d4, P=0.01, 1,106.0±659.0 vs. 2,858.8±141.3; d7, P=0.02, 761.8±746.2 vs. 2,357.3±304.3), and SF lactate (d7, P<0.0001, 5.4±0.2 vs. 15.0±0.3), SAA (endterm, P=0.01, 0.0 vs. 2,094.0±601.6), IL-6 (P=0.03, 313.0±119.2 vs. 1,328.2±208.9), and IL-18 (P=0.02, 11.1±0.5 vs. 13.3±3.8) were improved in TLR-MSC-VAN vs. VAN horses. Study limitations include the small horse sample size, short study duration, and lack of additional control groups.
UNASSIGNED: Combined TLR-activated MSC with antibiotic therapy may be a promising approach to manage joint infections with drug resistant bacteria.

Intra-articular microfragmented adipose tissue (MF-AT) injections have been proposed for the treatment of knee osteoarthritis (OA).
To compare a single injection of MF-AT or platelet-rich plasma (PRP) in terms of clinical outcomes and OA progression.
Randomized controlled trial; Level of evidence, 1.
A total of 118 patients with symptomatic knee OA were randomized to receive a single intra-articular injection of MF-AT or PRP. Patients were evaluated before the injection and at 1, 3, 6, 12, and 24 months with the International Knee Documentation Committee (IKDC) subjective score, Knee injury and Osteoarthritis Outcome Score (KOOS) subscales, EuroQol visual analogue scale (EQ-VAS), EuroQol 5 dimensions (EQ-5D), and visual analogue scale (VAS) for pain. Primary outcomes were the IKDC subjective score and the KOOS pain subscore at 6 months. Knees were evaluated at baseline and at 6, 12, and 24 months with radiography and high-resolution magnetic resonance imaging (MRI) using the Whole-Organ Magnetic Resonance Imaging Score (WORMS).
Both MF-AT and PRP provided a statistically and clinically significant improvement up to 24 months. The improvement in the IKDC subjective score from baseline to 6 months was similar in both MF-AT (41.1 ± 16.3 to 57.3 ± 18.8) and PRP (44.8 ± 17.3 to 58.4 ± 18.1) groups (P < .0005). The improvement in the KOOS pain subscore from baseline to 6 months was similar in both the MF-AT (58.4 ± 15.9 to 75.8 ± 17.4) and PRP (63.5 ± 17.8 to 75.5 ± 16.1) groups (P < .0005). Overall, no differences were found between the MF-AT and PRP groups in terms of clinical outcomes, adverse events (18.9% and 10.9%, respectively), and failures (15.1% and 25.5%, respectively). Radiographic and MRI findings did not show changes after the injection. As a secondary outcome, more patients in the MF-AT group with moderate/severe OA reached the minimal clinically important difference for the IKDC score at 6 months compared with the PRP group (75.0% vs 34.6%, respectively; P = .005).
A single intra-articular injection of MF-AT was not superior to PRP, with comparable low numbers of failures and adverse events and without disease progression. No differences were found in clinical and imaging results between the 2 biological approaches.

Hematopoietic stem cell transplantation (HSCT) is a standard therapy strategy for most malignant disorders in children. However, transplant-related pneumonia remains a major therapy challenge and mesenchymal stromal cells (MSCs) are rarely reported in HSCT-related pneumonia. The aim of our study was to assess the efficacy of MSC for HSCT-related pneumonia in children.
We retrospectively retrieved HSCT-related (severe and non-severe) pneumonia cases (aged < 18 years), which underwent MSC treatment (MSC group) or non-MSC treatment (non-MSC group) in Guangzhou Women and Children\'s Medical Center, from December 2017 to December 2019. We investigated outcomes of the two different treatments among severe cases and non-severe cases, respectively. The primary endpoints were differences in overall cure rate and time to cure between MSC and non-MSC groups. The secondary endpoints were 180-day overall survival and cumulative cure rate.
Finally, 31 severe pneumonia cases (16 in MSC group, 15 in non-MSC group) and 76 non-severe cases (31 in MSC group, 45 in non-MSC group) were enrolled in this study. Among severe pneumonia cases, overall cure rate in MSC group was significant higher than that in non-MSC group (12[75.0%] vs. 5[33.3%]; OR = 6.00, 95% CI [1.26-28.5]; p = 0.020); the time (days) to cure in MSC group was dramatically reduced compared with that in non-MSC group (36 [19-52] vs. 62 [42-81]; OR = 0.32, 95% CI [0.12-0.88]; p = 0.009); the 180-day overall survival in MSC group was better than that in non-MSC group (74.5% [45.4-89.6] vs. 33.3% [12.2-56.4]; p = 0.013). Among non-severe pneumonia cases, the time (days) to cure in MSC group was notably decreased compared with that in non-MSC group (28 [24-31] vs. 33 [26-39]; OR = 0.31, 95% CI [0.18-0.56]; p = 0.003). Compared with non-MSC group, MSC-treated patients achieved significant improvements of cumulative cure rate not only in severe pneumonia cases (p = 0.027), but also in non-severe cases (p < 0.001).
This study revealed that MSC treatment could contribute to improving outcomes in children with pneumonia post-HSCT, especially in severe cases. These findings suggest MSC treatment as a promising therapy for HSCT-related pneumonia in children.

Mesenchymal stromal cells (MSCs) have been shown to inhibit aerobic glycolysis in activated T cells, leading to increased autophagy. Although tryptophan depletion induced by indoleamine 2,3-dioxygenase (IDO) generated by MSCs has been suggested as a potential mechanism, we found that this inhibition was completely abolished when T cells were physically separated from MSCs using the Transwell system. Instead, in the current study, we demonstrate that programmed cell death 1 receptor (PD-1) and its ligand PD-L1, the expression of which is induced on activated T cells and MSCs, respectively, in response to IFN-γ are involved in this inhibition. Blockade of PD-1/PD-L1 interaction by blocking antibodies significantly restored glucose uptake, glycolytic activity, and cluster formation of activated T cells, whereas a specific inhibitor of IDO, 1-methyl-DL-tryptophan, had no effect. Neither surface nor cytoplasmic glucose transporter-1 expression on T cells was changed by MSCs. In addition, glycolytic gene expression in activated T cells was not inhibited despite the presence of MSCs. However, we found that hexokinase II (HK2) protein expression was markedly decreased in activated T cells that had been cocultured with MSCs. PD-1 blocking antibody restored HK2 expression. Taken together, our findings indicate that the PD-1/PD-L1 axis is involved in the MSC-mediated suppression of T cell glycolysis by negatively regulating HK2 activity at the protein level, but not at the mRNA level.

The ability of mesenchymal stromal cells (MSCs) to release a plethora of immunomodulatory factors makes them valuable candidates to overcome inflammatory bowel diseases (IBD). However, this cell therapy approach is still limited by major issues derived from nude MSC-administration, including a rapid loss of their immunomodulatory phenotype that impairs factor secretion, low persistence and impossibility to retrieve the cells in case of adverse effects. Here, we designed a licensing hydrogel system to address these limitations and thus, obtain a continuous delivery of bioactive factors. IFNγ-loaded heparin-coated beads were included in injectable in situ crosslinking alginate hydrogels, providing a 3D microenvironment that ensured continuous inflammatory licensing, cell persistence and implant retrievability. Licensing-hydrogel encapsulated human MSCs (hMSCs) were subcutaneously xenotransplanted in an acute mouse model of ulcerative colitis. Results showed that encapsulated hMSCs exerted a delocalized systemic protection, not presenting significant differences to healthy mice in the disease activity index, colon weight/length ratio and histological score. At day 7, cells were easily retrieved and ex vivo assays showed fully viable hMSCs that retained an immunomodulatory phenotype, as they continued secreting factors including PGE2 and Gal-9. Our data demonstrate the capacity of licensing hydrogel-encapsulated hMSCs to limit the in vivo progression of IBD.