Injection Of Mesenchymal Stromal Cells Research Articles

Functionality of bone marrow mesenchymal stromal cells derived from head and neck cancer patients – A FDA-IND enabling study regarding MSC-based treatments for radiation-induced xerostomia

PurposeSalivary dysfunction is a significant side effect of radiation therapy for head and neck cancer (HNC). Preliminary data suggests that mesenchymal stromal cells (MSCs) can improve salivary function. Whether MSCs from HNC patients who have completed chemoradiation are functionally similar to those from healthy patients is unknown. We performed a pilot clinical study to determine whether bone marrow-derived MSCs [MSC(M)] from HNC patients could be used for the treatment of RT-induced salivary dysfunction. MethodsAn IRB-approved pilot clinical study was undertaken on HNC patients with xerostomia who had completed treatment two or more years prior. Patients underwent iliac crest bone marrow aspirate and MSC(M) were isolated and cultured. Culture-expanded MSC(M) were stimulated with IFNγ and cryopreserved prior to reanimation and profiling for functional markers by flow cytometry and ELISA. MSC(M) were additionally injected into mice with radiation-induced xerostomia and the changes in salivary gland histology and salivary production were examined. ResultsA total of six subjects were enrolled. MSC(M) from all subjects were culture expanded to > 20 million cells in a median of 15.5 days (range 8–20 days). Flow cytometry confirmed that cultured cells from HNC patients were MSC(M). Functional flow cytometry demonstrated that these IFNγ-stimulated MSC(M) acquired an immunosuppressive phenotype. IFNγ-stimulated MSC(M) from HNC patients were found to express GDNF, WNT1, and R-spondin 1 as well as pro-angiogenesis and immunomodulatory cytokines. In mice, IFNγ-stimulated MSC(M) injection after radiation decreased the loss of acinar cells, decreased the formation of fibrosis, and increased salivary production. ConclusionsMSC (M) from previously treated HNC patients can be expanded for auto-transplantation and are functionally active. Furthermore IFNγ-stimulated MSC(M) express proteins implicated in salivary gland regeneration. This study provides preliminary data supporting the feasibility of using autologous MSC(M) from HNC patients to treat RT-induced salivary dysfunction

Preliminary evaluation of safety and migration of immune activated mesenchymal stromal cells administered by subconjunctival injection for equine recurrent uveitis.

Equine recurrent uveitis (ERU), an immune mediated disease characterized by repeated episodes of intra-ocular inflammation, affects 25% of horses in the USA and is the most common cause of glaucoma, cataracts, and blindness. Mesenchymal stromal cells (MSCs) have immunomodulatory properties, which are upregulated by preconditioning with toll-like receptor agonists. The objective was to evaluate safety and migration of TLR-3 agonist polyinosinic, polycytidylic acid (pIC)-activated MSCs injected subconjunctivally in healthy horses prior to clinical application in horses with ERU. We hypothesized that activated allogeneic MSCs injected subconjunctivally would not induce ocular or systemic inflammation and would remain in the conjunctiva for >14 days. Bulbar subconjunctiva of two horses was injected with 10 × 106 pIC-activated (10 μg/mL, 2 h) GFP-labeled MSCs from one donor three times at two-week intervals. Vehicle (saline) control was injected in the contralateral conjunctiva. Horses received physical and ophthalmic exams [slit lamp biomicroscopy, rebound tonometry, fundic examination, and semiquantitative preclinical ocular toxicology scoring (SPOTS)] every 1-3 days. Systemic inflammation was assessed via CBC, fibrinogen, and serum amyloid A (SAA). Horses were euthanized 14 days following final injection. Full necropsy and histopathology were performed to examine ocular tissues and 36 systemic organs for MSC presence via IVIS Spectrum. Anti-GFP immunohistochemistry was performed on ocular tissues. No change in physical examinations was noted. Bloodwork revealed fibrinogen 100-300 mg/dL (ref 100-400) and SAA 0-25 μg/mL (ref 0-20). Ocular effects of the subjconjucntival injection were similar between MSC and control eyes on SPOTS grading system, with conjunctival hypermia, chemosis and ocular discharge noted bilaterally, which improved without intervention within 14 days. All other ocular parameters were unaffected throughout the study. Necropsy and histopathology revealed no evidence of systemic inflammation. Ocular histopathology was similar between MSC and control eyes. Fluorescent imaging analysis did not locate MSCs. Immunohistochemistry did not identify intact MSCs in the conjunctiva, but GFP-labeled cellular components were present in conjunctival phagocytic cells. Allogeneic pIC-activated conjunctival MSC injections were well tolerated. GFP-labeled tracking identified MSC components phagocytosed by immune cells subconjunctivally. This preliminary safety and tracking information is critical towards advancing immune conditioned cellular therapies to clinical trials in horses.

Mesenchymal Stromal Cell Infusion Might Improve Bronchiolitis Obliterans after Allogeneic Hematopoietic Stem Cell Transplantation and Modulate Alveolar Macrophages in Mice

Some studies suggested that mesenchymal stromal cell (MSC) infusion might improve bronchiolitis obliterans (BO) in patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the underlying mechanism is little known. In this study, we investigated whether MSC infusion could improve BO after allo-HSCT in mice, and sought to explore its effects on lung immune microenvironment. To construct a BO after allo-HSCT model, bone marrow cells and spleen cells were harvested from C57BL/6 mice. BALB/c mice were lethally irradiated and received 5 × 10 6 bone marrow cells (for BM mice) or 5 × 10 6 bone marrow cells with 10 6 spleen cells (for BO mice) transplantation via tail vein injection. At day 28 post-transplantation, narrowing of the small airway and collagen accumulation within the small airway walls were observed in the BO mice instead of BM mice, as shown by Masson and hematoxylin-eosin staining. Bronchoalveolar lavage fluid (BALF) from the BO mice contained more lymphocytes and neutrophils than the BM mice. Also, autoreactive IgG was observed in bronchial epithelial cells in the BO group only. These data suggests that our model shared similar histological characteristics with BO patients. Alveolar macrophages (AMs) are a major immunocyte subgroup in the lung. The AMs of the BO mice had increased size compared to the BM mice, similar to “foam cells” in atherosclerosis. The number of “form cells” increased by time in the BO group, but was not found in the BM group. A significantly increased the transcription level of CD36 and ABCA1, two key lipid transport genes, was observed in the BO group at day 21, 30, and 45 post-transplantation. Also, mRNA level of M2-like macrophage-related genes like TGF-β1, CD206, Ym1 and Arg1 was upregulated significantly in the BO group compared to the BM group, since day 21 post-transplantation, but not TNF-α, a typical M1-related marker. We next tested could MSC infusion improve the outcomes of BO mice and its influences on the phenotype of AMs. To test this hypothesis, recipients were divided into four groups (BM, BO, MSC1 and MSC2). MSCs were isolated and cultured from femur bone chips of C57BL/6 mice. Mice within the MSC1 group received 10 6 MSCs via tail vein injection at day 7 and day 21 post-transplantation. Mice within the MSC2 group received 10 6 MSCs via tail vein injection at day 21 and day 28 post-transplantation. Mice within the BM or BO group did not receive MSC injection. BALF was harvested at day 14, 35, and 49. The cGVHD symptom score of the MSC1 group reached peak at day 21 post-transplantation and showed a trend of decrease afterwards. On the other hand, although the MSC2 group also showed the same trend, but increased again after day 42. The MSC1 group have significantly lower cGVHD symptom score compared to the BO group at day 35, 42 and 49. Also, the MSC1 group showed a trend of increased survival compared to the BO group. As for AM phenotypes, MSC1 group had a trend of decreased proportion on both M1-like (CD206- CD86+ iNOS+) and M2-like (CD86- CD163+) AMs compared to the BO group at day 14. At day 49, the MSC 1 and MSC2 group both showed a trend of decreased M2-like AMs and regulatory T cells (CD25+ Foxp3+) proportion. Taking together, we found that AMs of BO mice trended a M2-like phenotype. MSC infusion could improve the outcomes of BO mice, and modulate the phenotype of AMs.

Abstract 15869: Antiarrhythmic Effects of Mesenchymal Stromal Cells in a Post-Infarct Swine Model of Ventricular Tachycardia

Introduction: Mesenchymal Stromal Cells (MSC) reduce scar burden in models of ischemic cardiomyopathy. It is unknown whether MSCs exert antiarrhythmic effects. Hypothesis: MSCs reduce inducibility of monomorphic VT (mmVT) or prolong VT cycle length by ≥20 msec in a post-infarct swine model of VT. Methods: Two groups of 8 Yorkshire Swine underwent a 180-minute occlusion of the LAD. Group 1 (n=8) pigs underwent initial MRI scan, electrophysiology study (EPS), and 3D electroanatomic mapping (EAM) with injection of open-label MSCs (200M cells) around the borderzone of the infarct, using a NOGA Myostar catheter (J&J) guided by EAM. Group 1 pigs had repeat EAM, and final EPS 8 weeks later. Group 2 pigs (n=8) were randomized between placebo and MSC and underwent sequential MSC injections (100M cells/procedure), 7 weeks apart, followed by final EPS and MRI 7 weeks later. Scar% = total scar/myocardial mass (grams). Results: MmVT was induced in 5 of 8 (62.5%) at initial EPS (i-EPS) in Group 1 pigs and 3 of 8 (37.5%) at final EPS (f-EPS). In Group 2 all 4 MSC-treated swine had inducible mmVT during i-EPS but only 2 had mmVT at f-EPS. Overall, 9 of 12 MSC-treated pigs had mmVT initially and 5 of 12 pigs had mmVT at f-EPS (p=0.12). For MSC-treated pigs with persistently inducible mmVT (Groups 1 & 2), the baseline VT CL increased in all pigs by ≥20msec and increased from 216 ± 17 msec initially to 274 ± 35 msec at f-EPS (p = 0.001). In Group 1, Scar% was unchanged (22.2±1.6% to 20.5±2.1%) after single MSC treatment (p=0.17). In Group 2 MSC-treated pigs, Scar% decreased from 25.8±2.0% to 17.9±0.6% after 2 MSC treatments (p=0.014). In pigs with mmVT at i-EPS, the scar% was 24.4±1.2%, compared with 19.3±2.0% in pigs without i-EPS inducible mmVT (p=0.025). In pigs with mmVT at f-EPS, scar% was 22.5±1.8% compared with 18.1±1.6% in pigs without mmVT at f-EPS (p=0.048). Placebo-treated pigs showed no change in scar% (22.5±2.6% to 22.4±2.9%, p=0.44) and no change in VT inducibility (3 of 4 pigs at i-EPS and f-EPS). Conclusions: This study demonstrates that MSC injection reduced myocardial scar and either suppressed induction of VT or prolonged VT cycle length, consistent with an antiarrhythmic effect. Lower scar burden was associated with non-inducibility of VT, either at baseline or final EPS.

Comparative Effects of Intra-Articular versus Intravenous Mesenchymal Stromal Cells Therapy in a Rat Model of Osteoarthritis by Destabilization of Medial Meniscus.

Transplanted mesenchymal stromal cells (MSCs) exhibit a robust anti-inflammatory and homing capacity in response to high inflammatory signals, as observed in studies focused on rheumatic diseases that target articular cartilage (AC) health. However, AC degradation in osteoarthritis (OA) does not necessarily coincide with a highly inflammatory joint profile. Often, by the time patients seek medical attention, they already have damaged AC. In this study, we examined the therapeutic potential of a single bone marrow MSC transplant (2 × 106 cells/kgbw) through two different routes: intra-articular (MSCs-IAt) and intravenous (MSCs-IVt) in a preclinical model of low-grade inflammatory OA with an established AC degeneration. OA was induced through the destabilization of the medial meniscus (DMM) in female Wistar Kyoto rats. The animals received MSCs 9 weeks after surgery and were euthanized 4 and 12 weeks post-transplant. In vivo and ex vivo tracking of MSCs were analyzed via bioluminescence and imaging flow cytometry, respectively. Cytokine/chemokine modulation in serum and synovial fluid was measured using a multiplex panel. AC degeneration was quantified through histology, and hindlimb muscle balance was assessed with precision weighing. To our knowledge, we are the first group to show the in vivo (8 h) and ex vivo (12 h) homing of cells to the DMM-OA joint following MSCs-IVt. In the case of MSCs-IAt, the detection of cellular bioluminescence at the knee joint persisted for up to 1 week. Intriguingly, intra-articular saline injection (placebo-IAt) resulted in a worse prognosis of OA when compared to a non-invasive control (placebo-IVt) without joint injection. The systemic cytokines/chemokines profile exhibited a time-dependent variation between transplant routes, displaying a transient anti-inflammatory systemic response for both MSCs-IVt and MSCs-IAt. A single injection of MSCs, whether administered via the intra-articular or intravenous route, performed 9 weeks after DMM surgery, did not effectively inhibit AC degeneration when compared to a non-invasive control.

HISTOLOGICAL STRUCTURE OF THE RAT FEMURS AFTER FILLING OF DEFECTS IN THE DISTAL METAPHYSIS WITH 3D-PRINTED IMPLANTS BASED ON POLYLACTIDE AND TRICALCIUM PHOSPHATE IN COMBINATION WITH MESENCHYMAL STROMAL CELLS

Polylactide (PLA) frameworks printed on a 3D printer are used for filling the bone defects. The osteotropic properties of 3D-PLA can be improved by combining with tricalcium phosphate (TCP) and mesenchymal stromal cells (MSCs). Objective. Study the reconstruction in the rat femurs after implanting 3D-printed implants based on PLA and TCP (3D-I) in combination with cultured allogeneic MSCs into defects in the distal metaphysis. Methods. 48 white laboratory rats (age 5–6 months) were used, which were randomly divided into groups: Control — 3D-I; Experiment-I — 3D-I, saturated MSCs; Experiment II — 3D-I, with injection of 0.1‒0.2 ml of medium with MSCs into the area of surgical intervention 7 days after implantation. 15, 30 and 90 days after the operation, histological (with histomorphometry) studies were conducted. Results. The area of 3D-I decreased with time in all groups and connective and bone tissues formed in different ratios. 15 days after the surgery, in the Experiment-I group, the area of the connective tissue was 1.9 and 1.6 times greater (p<0.001) in comparison to the Control and Experiment II; 30 days it was greater 1.6 times (p < 0.001) and 1.4 times (p=0.001), respectively. 30 days after the surgery, the area of newly formed bone in the Experiment-I group was 2.2 times (p < 0.001) less than in the Control. On the contrary, in the Experiment-II, the area of newly formed bone was 1.5 and 3.3 times greater (p < 0.001) compared to Experiment-I and Control, respectively. Conclusions. The studied 3D-I with time after their implantation into the metaphyseal defects of the rats’ femurs are replaced by connective and bone tissues. The use of 3D-I, saturated MSCs, 15 and 30 days after the surgery, caused excessive formation of connective tissue and slower bone formation. Local injection of MSCs 7 days after the implantation of 3D-I caused to the formation of a larger area of newly bone 30th day after surgery compared to 3D-I alone and 3D-I with MSCs.

A Pilot, First in Human Study of Autologous IFN-Gamma Stimulated Mesenchymal Stromal Cells for Treatment of Radiation-Induced Xerostomia

There are no existing effective treatments for radiation-induced xerostomia (RIX), a common side effect of head and neck radiation. Mesenchymal stromal cells (MSCs) exhibit regenerative effects in multiple tissues and may represent an effective cell therapy for the treatment of RIX. Here we present the primary safety and secondary efficacy endpoints of a first-in-human pilot study of IFNγ-stimulated autologous bone marrow- derived MSCs [MSC(M)] for the treatment of RIX. We conducted a single-center clinical trial investigating the safety and tolerability of autologous IFNγ-stimulated MSC(M). The study was conducted under an FDA-IND and approved by the local IRB. Patients underwent bone marrow aspiration, MSC(M) were then culture-expanded, stimulated with IFNγ, and cryopreserved. Banked IFNγ-stimulated MSC(M) were thawed, allowed to recover, and then 10 × 106 MSC(M) were injected transcutaneously via ultrasound guidance into one submandibular gland. The primary objective was safety and tolerability determined by dose-limiting toxicity (DLT) defined as submandibular pain > 5 on a standard 10-point pain scale or any serious adverse event (SAE) within one month after injection. Secondary objectives included analysis of efficacy as measured by salivary quantification and using 3 validated quality of life instruments. Quantitative results are reported as mean and standard deviation (SD). Six radiation-induced xerostomia patients with head and neck cancer who had completed radiation at least 2 years earlier were enrolled. The median age was 71 (61-74) and 5 (83%) patients were male. Five patients (83%) were treated with chemoradiation and one patient (17%) with radiation alone. The average dose of radiation to the injected submandibular gland was 59.9 Gy. Three patients (50%) reported a pain score of 1 after submandibular gland injection, all pain resolved within 4 days. No patients reported pain 1 month after injection, with no SAEs or other DLTs reported 1 month after injection. The analysis of secondary endpoints demonstrated a trend of increased salivary production. The mean unstimulated saliva was 0.13 mL/min (SD 0.17) at baseline and increased to 0.14 mL/min (SD 0.12) at 1 month after injection and 0.19 mL/min (SD 0.21) at 3-months. Quality of life surveys also showed a trend towards improvement. Injection of autologous IFNγ-stimulated MSC(M) into the submandibular gland of patients with RIX is safe and well tolerated. A trend towards an improvement in secondary endpoints of salivary quantity and quality of life was observed. This first-in-human pilot study provides support for further investigation into IFNγ-stimulated MSC(M) as an innovative, potentially curative, remedy to treat RIX. A phase I dose-escalation study injecting into bilateral submandibular glands is scheduled to begin accrual in the spring of 2023.

CHANGES IN MARKERS OF BONE TISSUE REMODELING AND THE INFLAMMATORY PROCESS IN THE BLOOD SERUM OF WHITE RATS IN CASE OF DEFECT FILLING OF THE FEMUR WITH IMPLANTS BASED ON POLYLACTIDE AND TRICALCIUMPHOSPHATE WITH MESENCHYMAL STEM CELLS

Objective. Based on the analysis of markers of inflammation and metabolism of bone tissue in the blood serum of laboratory rats, to evaluate the course of bone remodeling after filling the defect in the distal metaphysis of the femur with 3D-printed implants based on polylactide and tricalcium phosphate (3D-I) alone or in combination with mesenchymal stromal cells (MSCs). Methods. 53 white rats were used, which were divided into groups: intact (5 animals) — the operation was not performed; Control (15) — 3D-I; Experiment I (15) — 3D-I + cultured alloMSCs; Experiment II (15) — 3D-I + introduction of alloMSCs into the area of surgical intervention 7 days after implantation. The following were studied: the content of glycoproteins (GP), interleukin-6 (IL-6), osteocalcin, chondroitin sulfates (CS), total protein, calcium, alkaline (AlP) and acid phosphatase (AP) activity, and their ratio, mineralization indices were calculated. Results. Compared with intact animals, higher indicators were determined in the rats of the Control group: the content of GP by 39.73; 32.88; 23.29 %; CS — 250.00; 222.09 and 196.51 %, AlP activity — 81.67, 51.03, 39.36 %, on the 15th, 30th, and 90th days of the experiment; IL-6 — 44.89; 60.06 % on the 15th and 30th days. In the rats of the Experiment I g roup: the content of GP — by 82.19; 65.75, 57.53 %, IL-6 — 72.14; 96.59; 79.88 %, CS — 306.98; 276.74; 253.49 %; AlP activity — 63.73; 129.70; 51.28 %, on the 15th, 30th and 90th days of the experiment. In the Experiment II group: on the 15th, 30th and 90th days, the content of GP was higher by 27.40; 26.03; 129.18 %; CS — by 175.58; 137.21 and 115.12 %; AlP activity — 192.99; 178.02, 76.31 %; on the 15th and 30th days: IL-6 — by 37.46; 20.74 %. Conclusions. In the case of filling the defect with 3D-printed implants, biochemical signs of moderate inflammation were determined; 3D-printed implants together with MSCs — pronounced inflammation, slowing of bone formation, formation of connective tissue; 3D-printed implants with postoperative injection of MSCs — moderate inflammation and optimal conditions for healing the defect with bone tissue.

Allogenic mesenchymal stromal cells and their extracellular vesicles in COVID-19 induced ARDS: a randomized controlled trial

Background and aimsThe main causes of death in patients with severe Coronavirus disease-2019 (COVID-19) are acute respiratory distress syndrome (ARDS) and multiorgan failure caused by a severe inflammatory cascade. Novel treatment strategies, such as stem-cell-based therapy and their derivatives can be used to relieve inflammation in these cases. In this study, we aimed to evaluate the safety and efficacy of therapy using mesenchymal stromal cells (MSCs) and their derived extracellular vesicles in COVID-19 patients.Materials and methodsCOVID-19 patients with ARDS were included in this study and allocated into two study and control groups using block randomization. While all patients received recommended treatment based on guidelines from the national advisory committee for COVID-19 pandemic, the two intervention groups received two consecutive injections of MSCs (100 × 106 cells) or one dose of MSCs (100 × 106 cells) followed by one dose of MSC-derived extracellular vesicles (EVs). Patients were assessed for safety and efficacy by evaluating clinical symptoms, laboratory parameters, and inflammatory markers at baseline and 48 h after the second intervention.ResultsA total number of 43 patients (the MSC alone group = 11, MSC plus EV group = 8, and control group = 24) were included in the final analysis. Mortality was reported in three patients in the MSC alone group (RR: 0.49; 95% CI 0.14–1.11; P = 0.08); zero patient in the MSC plus EV group (RR: 0.08; 95% CI 0.005–1.26; P = 0.07) and eight patients in the control group. MSC infusion was associated with a decrease in inflammatory cytokines such as IL-6 (P = 0.015), TNF-α (P = 0.034), IFN-γ (P = 0.024), and CRP (P = 0.041).ConclusionMSCs and their extracellular vesicles can significantly reduce the serum levels of inflammatory markers in COVID-19 patients, with no serious adverse events.Trial registration IRCT, IRCT registration number: IRCT20200217046526N2. Registered 13th April 2020, http://www.irct.ir/trial/47073.

Cartilage regeneration and inflammation modulation in knee osteoarthritis following injection of allogeneic adipose-derived mesenchymal stromal cells: a phase II, triple-blinded, placebo controlled, randomized trial

BackgroundIntra-articular injection of mesenchymal stromal cells (MSCs) with immunomodulatory features and their paracrine secretion of regenerative factors proposed a noninvasive therapeutic modality for cartilage regeneration in knee osteoarthritis (KOA).MethodsTotal number of 40 patients with KOA enrolled in two groups. Twenty patients received intra-articular injection of 100 × 106 allogeneic adipose-derived mesenchymal stromal cells (AD-MSCs), and 20 patients as control group received placebo (normal saline). Questionnaire-based measurements, certain serum biomarkers, and some cell surface markers were evaluated for 1 year. Magnetic resonance imaging (MRI) before and 1 year after injection was performed to measure possible changes in the articular cartilage.ResultsForty patients allocated including 4 men (10%) and 36 women (90%) with average age of 56.1 ± 7.2 years in control group and 52.8 ± 7.5 years in AD-MSCs group. Four patients (two patients from AD-MSCs group and two patients from the control group) excluded during the study. Clinical outcome measures showed improvement in AD-MSCs group. Hyaluronic acid and cartilage oligomeric matrix protein levels in blood serum decreased significantly in patients who received AD-MSCs (P < 0.05). Although IL-10 level significantly increased after 1 week (P < 0.05), the serum level of inflammatory markers dramatically decreased after 3 months (P < 0.001). Expressions of CD3, CD4, and CD8 have a decreasing trend during 6-month follow-up (P < 0.05), (P < 0.001), and (P < 0.001), respectively. However, the number of CD25+ cells increased remarkably in the treatment group 3 months after intervention (P < 0.005). MRI findings showed a slight increase in the thickness of tibial and femoral articular cartilages in AD-MSCs group. The changes were significant in the medial posterior and medial anterior areas of ​​the tibia with P < 0.01 and P < 0.05, respectively.ConclusionInter-articular injection of AD-MSCs in patients with KOA is safe. Laboratory data, MRI findings, and clinical examination of patients at different time points showed notable articular cartilage regeneration and significant improvement in the treatment group.Trial registration: Iranian registry of clinical trials (IRCT, https://en.irct.ir/trial/46), IRCT20080728001031N23. Registered 24 April 2018.

Understanding the effects of mesenchymal stromal cell therapy for treating osteoarthritis using an in vitro co-culture model.

Osteoarthritis (OA) is a leading cause of chronic pain and disability, for which there is no cure. Mesenchymal stromal cells (MSCs) have been used in clinical trials for treating OA due to their unique ability to generate paracrine anti-inflammatory and trophic signals. Interestingly, these studies have shown mainly short-term effects of MSCs in improving pain and joint function, rather than sustained and consistent benefits. This may reflect a change or loss in the therapeutic effects of MSCs after intra-articular injection. The present study aimed to unravel the reasons behind the variable efficacy of MSC injections for OA using an in vitro co-culture model. Osteoarthritic human synovial fibroblasts (OA-HSFs) were co-cultured with MSCs to investigate their reciprocal effects on cell responses and whether a short-term exposure of OA cells to MSCs was sufficient for reducing their diseased characteristics in a sustained manner. Gene expression and histological analyses were performed. OA-HSFs exposed to MSCs showed short-term downregulation of inflammatory markers. However, the MSCs showed upregulation of inflammatory markers and impaired ability to undergo osteogenesis and chondrogenesis in the presence of OA-HSFs. Moreover, short-term exposure of OA-HSFs to MSCs was found to be insufficient for inducing sustained changes to their diseased behaviour. These findings suggested that MSCs may not provide long-term effects in correcting the OA joint environment due to them adopting the diseased phenotype of the surrounding tissues, which has important implications for the future development of effective stem-cell-based OA treatments with long-term therapeutic efficacy.

USING MESENCHYMAL STROMAL CELLS AND THEIR SECRETOME TO IMPROVE OSTEOCHONDRAL REPAIR IN A MURINE MODEL

Osteochondral injuries are a recognised factor in the development of osteoarthritis (OA). Mesenchymal stromal cells (MSCs) represent a promising biological therapeutic option as an OA-modifying treatment, and they also secrete factors that may have an anti-catabolic effect and/or encourage endogenous repair. We aim to study the effects of (i) intra-articular injection of human bone-marrow-derived MSCs and (ii) their secretome on recovery in a murine knee osteochondral injury model.The MSC secretome was generated by stimulating human bone-marrow-derived MSCs with tumour necrosis factor alpha (TNFα). Mice (n=48) were injected with i) MSC secretome, ii) MSCs or iii) cell culture medium (control). Pain was assessed by activity monitoring, and cartilage repair, subchondral bone volume and synovial inflammation were evaluated using histology and microCT.Both MSC- and MSC-secretome-injected mice showed significant pain reduction at day 7 when compared to control mice, but only the MSC-injected mice maintained a significant improvement over the controls at day 28. Cartilage repair was significantly improved in MSC-injected mice. No significant effects were observed with regards to synovial inflammation or subchondral bone volume.The MSC secretome demonstrates regenerative effects but this does not appear to be as sustained as a MSC cell therapy. Further studies are required to investigate if this can be overcome using different dosing regiments for injection of the MSC secretome. As we further understand the regenerative properties of the MSC secretome, we may be able to enhance the clinical translatability of these therapies. Direct intra-articular injection of MSCs for the treatment of OA also appears promising as a potential future strategy for OA management.Acknowledgements: MS is supported by a grant from the Wellcome Trust (PhD Programme for Clinicians)

Stem cell-based interventions for the prevention and treatment of intraventricular haemorrhage and encephalopathy of prematurity in preterm infants.

Germinal matrix-intraventricular haemorrhage (GMH-IVH) and encephalopathy of prematurity (EoP) remain substantial issues in neonatal intensive care units worldwide. Current therapies to prevent or treat these conditions are limited. Stem cell-based therapies offer a potential therapeutic approach to repair, restore, or regenerate injured brain tissue. These preclinical findings have now culminated in ongoing human neonatal studies. This is an update of the 2019 review, which did not include EoP. To evaluate the benefits and harms of stem cell-based interventions for prevention or treatment of GM-IVH and EoP in preterm infants. We used standard, extensive Cochrane search methods. The latest search was April 2022. We attempted to include randomised controlled trials, quasi-randomised controlled trials, and cluster trials comparing 1. stem cell-based interventions versus control; 2. mesenchymal stromal cells (MSCs) of type or source versus MSCs of other type or source; 3. stem cell-based interventions other than MSCs of type or source versus stem cell-based interventions other than MSCs of other type or source; or 4. MSCs versus stem cell-based interventions other than MSCs. For prevention studies, we included extremely preterm infants (less than 28 weeks' gestation), 24 hours of age or less, without ultrasound diagnosis of GM-IVH or EoP; for treatment studies, we included preterm infants (less than 37 weeks' gestation), of any postnatal age, with ultrasound diagnosis of GM-IVH or with EoP. We used standard Cochrane methods. Our primary outcomes were 1. all-cause neonatal mortality, 2. major neurodevelopmental disability, 3. GM-IVH, 4. EoP, and 5. extension of pre-existing non-severe GM-IVH or EoP. We planned to use GRADE to assess certainty of evidence for each outcome. We identified no studies that met our inclusion criteria. Three studies are currently registered and ongoing. Phase 1 trials are described in the 'Excluded studies' section. No evidence is currently available to evaluate the benefits and harms of stem cell-based interventions for treatment or prevention of GM-IVH or EoP in preterm infants. We identified three ongoing studies, with a sample size range from 20 to 200. In two studies, autologous cord blood mononuclear cells will be administered to extremely preterm infants via the intravenous route; in one, intracerebroventricular injection of MSCs will be administered to preterm infants up to 34 weeks' gestational age.

Barriers to mesenchymal stromal cells for low back pain

Intervertebral disc degeneration is the main cause of low back pain. In the past 20 years, the injection of mesenchymal stromal cells (MSCs) into the nucleus pulposus of the degenerative disc has become the main approach for the treatment of low back pain. Despite the progress made in this field, there are still many barriers to overcome. First, intervertebral disc is a highly complex load-bearing composite tissue composed of annulus fibrosus, nucleus pulposus and cartilaginous endplates. Any structural damage will change its overall biomechanical function, thereby causing progressive degeneration of the entire intervertebral disc. Therefore, MSC-based treatment strategies should not only target the degenerated nucleus pulposus but also include degenerated annulus fibrosus or cartilaginous endplates. Second, to date, there has been relatively little research on the basic biology of annulus fibrosus and cartilaginous endplates, although their pathological changes such as annular tears or fissures, Modic changes, or Schmorl's nodes are more commonly associated with low back pain. Given the high complexity of the structure and composition of the annulus fibrosus and cartilaginous endplates, it remains an open question whether any regeneration techniques are available to achieve their restorative regeneration. Finally, due to the harsh microenvironment of the degenerated intervertebral disc, the delivered MSCs die quickly. Taken together, current MSC-based regenerative medicine therapies to regenerate the entire disc complex by targeting the degenerated nucleus pulposus alone are unlikely to be successful.

The ocular graft-versus-host disease: the path from current knowledge to future managements.

Graft-versus-host disease (GVHD) is one of the major complications of allogenic haematopoietic stem cell transplantation (HSCT). The manifestation of ocular GVHD (oGVHD) is variable and may involve cornea, lacrimal gland, conjunctiva, eyelid, and/or nasolacrimal duct. We reviewed and summarized the current managements of oGVHD with specific focus on the emerging therapeutic advances. PubMed, Web of Science, and Google Scholar were searched for relevant literatures published within 20 years. Keywords used included "Graft-Versus-Host Disease", "GVHD", "ocular", "ocular surface", "ocular GVHD", "oGVHD", "dry eye", "keratitis", etc. RESULTS: Current managements of oGVHD can be classified into topical immunosuppressants, local tear-preservatory treatments, local non-pharmacological/surgical interventions, and systemic treatments. Additionally, some innovative therapies with promising treatment effects have been proposed, including topical target therapies, epitheliotrophic and neurotrophic treatments, recombinant DNase eye drops, mesenchymal stromal cell injection, and more. Clinical managements of oGVHD are administered in a symptom-based, stepwise manner. The advances in innovative therapies may help improve clinical outcomes, and it is essential that physicians stay updated with these novel treatment options.

Activation of the kynurenine-aryl hydrocarbon receptor axis impairs the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells in osteoarthritis rats.

It has been proven that intra-articular injection of mesenchymal stromal cells (MSCs) can alleviate cartilage damage in osteoarthritis (OA) by differentiating into chondrocytes and protecting inherent cartilage. However, the mechanism by which the OA articular microenvironment affects MSCs' therapeutic efficiency is yet to be fully elucidated. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in various cellular processes, such as osteogenesis and immune regulation. Tryptophan (Trp) metabolites, most of which are endogenous ligand for AHR, are abnormally increased in synovial fluid (SF) of OA and rheumatoid arthritis (RA) patients. In this study, the effects of kynurenine (KYN), one of the most important metabolites of Trp, were evaluated on the chondrogenic and chondroprotective effects of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). hUC-MSCs were cultured in conditioned medium containing different proportions of OA/RA SF, or stimulated with KYN directly, and then, AHR activation, proliferation, and chondrogenesis of hUC-MSCs were measured. Moreover, the chondroprotective efficiency of short hairpin-AHR-UC-MSC (shAHR-UC-MSC) was determined in a rat surgical OA model (right hind joint). OA SF could activate AHR signaling in hUC-MSCs in a concentration-dependent manner and inhibit the chondrogenic differentiation and proliferation ability of hUC-MSCs. Similar results were observed in hUC-MSCs stimulated with KYN in vitro. Notably, shAHR-UC-MSC exhibited superior therapeutic efficiency in OA rat upon intra-articular injection. Taken together, this study indicates that OA articular microenvironment is not conducive to the therapeutic effect of hUC-MSCs, which is related to the activation of the AHR pathway by tryptophan metabolites, and thus impairs the chondrogenic and chondroprotective effects of hUC-MSCs. AHR might be a promising modification target for further improving the therapeutic efficacy of hUC-MSCs on treatment of cartilage-related diseases such as OA.

First experience in usage of lipoaspirate-derived mesenchymal stromal cells in combined treatment of the patients with liver cirrhosis

Liver cirrhosis (LC) is a serious problem with respect to drug therapy. Certain promises are associated with development of stem cell-based technologies, in particular, mesenchymal stromal cells (MSCs). Over recent years, a significant interest was drawn to the usage of MSCs isolated from adipose tissue, which have a number of advantages over bone marrow MSCs. The aim of this study was to evaluate safety and tolerability of transplantation of autologous MSCs derived from adipose tissue (lipoaspirate) when treating the patients with LC of different origin. The study group consisted of 12 patients (4 men and 8 women), aged 33 to 67 years. In six patients, development of LC was due to viral hepatitis; in other subjects it was caused by non-viral factors, including toxic effects (n = 3), primary biliary cirrhosis (n = 2) and cryptogenic cirrhosis (n = 1). The LC severity assessed by the Child-Pugh scale corresponded to class A (6 cases) or class B (6 patients). The MELD scores varied from 8 to 11. All patients were injected once with autologous MSCs (a mean of 12.94 106, 11.3 to 16.0 106) obtained by culturing the lipoaspirate cells for 14 days. Lipoaspiration and injection of MSCs were not accompanied by development of side effects, or serious adverse events. Analysis of clinical efficacy, carried out 6-12 months after therapy, made it possible to distinguish two subgroups: responders to the therapy (subgroup 1, n = 6) and non-responders (subgroup 2, n = 6). In subgroup 1, the MELD score was found to be decreased in 4 cases, and did not change in 2 patients. In contrast, the MELD score increased in the patients from subgroup 2. Both subgroups did not differ in etiology and severity of LC, or concomitant pathology (COVID-19). Positive dynamics in subgroup 1 was revealed for the signs of hepatocellular insufficiency (n = 1), cytolysis (n = 2) and cholestasis (n = 1). In two patients with MELD score stabilization, the scores reached the reference values. After MSC therapy, the majority of patients (8/12) underwent a new coronavirus infection, and the COVID incidence rate was similar in groups with or without response to MSC therapy (4 patients in each group). The results of the study suggest the safety of using MSCs isolated from the adipose tissue of patients with liver cirrhosis, and the perspectives of such approach in order to stabilize or improve liver function.

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.

Mesenchymal stromal cell therapy for COVID-19-induced ARDS patients: a successful phase 1, control-placebo group, clinical trial

BackgroundAcute respiratory distress syndrome (ARDS) is the devastating complication of the new COVID-19 pandemic, directly correlated with releasing large amounts of inflammatory cytokines. Due to their immunoregulatory features, mesenchymal stromal cells (MSCs) provide a promising approach against this disease. In this regard, this study was designed as a single-center, open-label, phase 1 clinical trial with a control group to examine the safety and explore the possible potency of three injections of umbilical cord-derived MSCs (UC-MSCs) in mild–moderate COVID-19-induced ARDS patients.MethodsTwenty confirmed COVID-19 patients with mild-to-moderate ARDS degree entered the study and were divided into two groups: control group (standard care) and intervention group (standard care + UC-MSCs). The patients received three intravenous infusions of UC-MSCs (1 × {10}^{6} cells/kg BW per injection) every other day. Respiratory markers, CRP levels and specific serum cytokines were assessed four times (days of 0, 5, 10 and 17) during the 17-day follow-up period.ResultsDuring the study, there were no serious adverse effects after cell transplantations. Besides, significant improvement in SPO2/FIO2 ratio and serum CRP levels was observed. On the other hand, a significant decrease (P < 0.05) in serum cytokine levels of IL-6, IFN-g, TNF-α, IL-17 A and a significant increase in serum cytokine levels of TGF-B, IL-1B and IL-10 were observed. Also, no significant changes were observed in CT scan images of patients during the study period.ConclusionOur obtained results demonstrated that multiple intravenous transplantations of allogenic UC-MSCs in non-severe COVID-19-induced ARDS patients are a safe procedure. In addition, this intervention is a hopeful approach to decline cytokine storm and recover respiratory functions. Indeed, more clinical trials with larger sample sizes are required to confirm these results.Trial registration This clinical trial was registered with the Iranian Registry of Clinical Trials (ID: IRCT20160809029275N1 at 2020.05.30).

Mesenchymal Stromal Cells Combined With Elastin-Like Recombinamers Increase Angiogenesis In Vivo After Hindlimb Ischemia.

Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)–based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti–human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti–CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.