Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics.

Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics.

A diet rich in omega-3 fatty acid improves periodontitis and tissue destruction by MMP2- and MMP9-linked inflammation in a murine model.

Periodontitis is an oral-cavity inflammatory disease and is the principal cause associated with tooth loss. Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are important proteases involved in periodontal tissue destruction. The omega-3 polyunsaturated fatty acids (ω-3 PUFA) have been demonstrated to possess immunoregulatory properties in periodontitis. The aim of the study was to investigate the effects of ω-3 PUFA on inflammation and on the expression of MMP-2 and -9 in a murine periodontitis model. Twenty-four male C57BL/6 mice were divided into control mice (Control), control mice treated with ω-3 PUFA (O3), mice with periodontitis (P), and mice with periodontitis treated with ω-3 PUFA (P + O3). ω-3 PUFA were administered orally once a day for 70 days. Periodontitis in mice was induced by Porphyromonas gingivalis-infected ligature placement around the second maxillary molar. The mice were sacrificed, and blood and maxillary samples were collected. Flow cytometry was used to quantify tumor necrosis factor-alpha (TNFα), interleukin (IL)-2, IL-4, IL-5, and interferon-gamma. Histologic analysis and immunohistochemistry for MMP-2 and -9 were performed. The data were statistically evaluated using analysis of variance (ANOVA) and the Tukey post hoc test. Histological analysis showed that ω-3 PUFA supplementation prevented inflammation and tissue destruction and revealed that bone destruction was more extensive in the P group than in the P + O3 group (p < 0.05). Also, it decreased the serum expressions of TNFα and IL-2 and the tissue expression of MMP-2 and -9 in the periodontitis-induced model (p < 0.05). ω-3 PUFA supplementation prevented alveolar bone loss and periodontal destruction, probably by decreasing the expression of MMP-2 and MMP-9 and its immunoregulatory properties.

Exercise improves intestinal IgA production by T-dependent cell pathway in adults but not in aged mice.

Introduction: Hypermutated high-affinity immunoglobulin A (IgA), neutralizes toxins and drives the diversification of bacteria communities to maintain intestinal homeostasis although the mechanism underlies the impact of moderate aerobic exercise (MAE) on the IgA-generation via T-dependent (TD) is not fully know. Therefore, the aim of this study was to determine the effect of long-time MAE on the production of IgA through the TD pathway in Peyer´s patches of the small intestine from aged mice. Methods: MAE protocol consisted of twenty 3-month-old (young) BALB/c mice running in an endless band at 0° inclination and a speed of 10 m/h for 5 days a week and resting 2 days on the weekend until reaching 6-month-old (adulthood, n=10) or 24-month-old (aging, n=10). Groups of young, adult, or elderly mice were included as sedentary controls (n=10/per group). At 6 or 24 months old, all were sacrificed, and small intestine samples were dissected to prepare intestinal lavages for IgA quantitation by ELISA and to obtain suspensions from Peyer´s patches (PP) and lamina propria (LP) cells for analysis of T, B, and plasma cell subpopulations by flow cytometry and mRNA analysis expression by RT-qPCR of molecular factors related to differentiation of B cells to IgA+ plasma cells, class switch recombination, and IgA-synthesis. Statistical analysis was computed with two-way ANOVA (factor A=age, factor B=group) and p<0.05 was considered for statistically significant differences. Results: Compared to age-matched sedentary control, in exercised elderly mice, parameters were either increased (IgA concentration, IL-21, IL-10 and RDH mRNA expression), decreased (α-chain mRNA, B cells, mIgA+ B cells, mIgM+ B cells and IL-4 mRNA) or unchanged (PP mIgA+ plasmablasts and LP cyt-IgA+ plasma cells). Regarding the exercised adult mice, they showed an up-modulation of IgA-concentration, mRNA expression IL-21, IL-10, and RDH and cells (PP B and T cells, mIgM+ plasmablasts and LP cyt-IgA+plasma cells). Conclusion: Our findings suggest that MAE restored the IgA production in adult mice via the TD cell pathway but does not in aged mice. Other studies are necessary to know in more detail the impact of long-time MAE on the TD pathway to produce IgA in aging.

[Peritoneal dialysis impact on quality of life in elderly]. / Impacto de la diálisis peritoneal en la calidad de vida de adultos mayores.

Background: Nowadays elderly live longer but with more diseases and geriatric syndromes which can deteriorate their quality of life (QoL). Peritoneal dialysis (PD) is a renal replacement therapy which seeks to prolong an improve QoL; however, this is uncertain in elderly. Therefore, comparing QoL before and after starting dialysis in this population may let us know if there is a benefit at this level. Objective: Identify the QoL that patients have before and after PD. Material and methods: Longitudinal, comparative, prospective cohort, before and after study. Elderly with End Stage Renal Disease in whom hospitalization for PD was indicated. QoL was measured the instrument KDQOL SF 1.3. before and after 2 months of PD. Statistical Analysis: T paired test was performed with the basal value of QoL and after. Risks with 95% confidence intervals and X2 were obtained between the basal characteristics and the dependent variable of QoL. Results: 21 patients. After 2 months the QoL had an increment in comparison to basal QoL, but with no statistical significance (63.47 [SD 16.63] Vs 56.83 [16.01], P= 0.22. In the 7th decade PD increased QoL by 13.01 points (P= 0.04). Conclusions: PD increases QoL in the 7th decade.

Introducción: en la actualidad, los adultos mayores (AM) viven más años, pero con más enfermedades y síndromes geriátricos, lo cual puede deteriorar su calidad de vida (CV). La diálisis peritoneal (DP) es una terapia de sustitución renal que se pretende mejorar la esperanza y la CV, aunque esto es incierto en los AM. Por lo tanto, comparar la CV antes y después de iniciar la DP en esta población permite saber si existe un beneficio a ese nivel. Objetivo: identificar la CV con la que cuentan los AM antes y después de DP. Material y métodos: cohorte prospectiva, comparativa, tipo antes y después en AM con enfermedad renal terminal quienes se hospitalizaron para iniciar DP. La CV de determinó con el instrumento KDQOL SF 1.3, antes y dos meses después de la DP. El análisis estadístico consistió en t pareada entre el puntaje de CV basal y después. Entre las características basales y la variable CV se obtuvieron riesgos con intervalos de confianza al 95%, así como Chi cuadrada. Se tomó como significativa una p bilateral de ≤ 0.05. Resultados: 21 pacientes. Luego de 2 meses iniciada la DP, el valor promedio de la CV mostró un incremento en comparación con la CV basal, aunque no logra significancia estadística (63.47 [DE: 16.63] frente a 56.83 [DE: 16.01], p = 0.22. En la séptima década de la vida, la DP produjo un incremento de 13.01 puntos en la CV (p = 0.04). Conclusiones: la DP mejora la CV en AM de la séptima década de la vida.

Bone Marrow as a Therapeutic Target for Type 2 Diabetes Complications.

Type 2 diabetes mellitus (T2DM) is a world epidemic with a high prevalence and mortality. The origin of macro and microvascular complications associated with T2DM is complex and new mechanisms to explain their development are emerging. The changes induced by T2DM in the microenvironment of bone marrow (BM) alter the expansion and differentiation of stem cells and have been related to the development of micro and macrovascular diseases. Alterations in the differentiation and function of hematopoietic, endothelial, and mesenchymal stem cells in T2DM patients reduced the mobility of BM stem cells to the circulation and some immature, dysfunctional, or inflammatory cells pass to the blood (mobilopathy). Consequently, tissue repair is impaired, and the tissue damage caused by hyperglycemia, oxidative stress, and inflammation is increased. These alterations can contribute to diabetic complications, decreasing the quality of life, and increasing mortality. The modulation of the bone marrow microenvironment may be a therapeutic target for treating T2DM and its complications. This article analyses the changes induced in BM and their impact on the development of cardiovascular and kidney complications in T2DM. Also, different therapeutic strategies to restore the bone marrow microenvironment and function through the modulation of oxidative stress, inflammation, and adipogenicity are discussed, considering bone marrow as a novel potential therapeutic target to treat vascular complications of diabetes.

Regionalized analysis of gut barrier components in the small intestine of BALB/c mice following chronic immobilization stress.

BACKGROUND: Microbiota and tight junction proteins (TJPs) are components of the gut barrier, and are considered stress targets that have deleterious effects on intestinal homeostasis. OBJECTIVES: This study aimed to evaluate the effects of chronic immobilization stress on selected small intestine homeostasis parameters. MATERIAL AND METHODS: Female BALB/c mice were divided into a stress group that underwent short-term immobilization for 2 h per day for 4 consecutive days, and a non-stressed control group (n = 6 per group). Proximal and distal small intestine samples were excised to assess colony-forming units per gram (CFU/g) of total bifidobacteria in selective agar plates, luminal albumin was assessed using immune-enzymatic assay, pro-inflammatory cytokines were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TJPs (pore-forming, claudin (Cld)-2; pore-sealing, Cld-4; ambiguous, Cld-7, -12 and -15) were assessed with RT-qPCR and western blotting. RESULTS: Compared with the control group, the stress group had lower body weight and energy intake. In the distal region, the stressed mice had lower bifidobacteria count and messenger ribonucleic acid (mRNA) expression of Cld-2, Cld-4 and Cld-12, though they had more albumin and higher interleukin (IL)-6 mRNA expression. Within the proximal region, the stressed mice had higher mRNA expression of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-6, Cld-7, Cld-12, and Cld-15, along with lower levels of IL-10 and Cld-4. However, mRNA and protein expression of TJPs were discordant. CONCLUSIONS: These findings indicate divergent stress-induced outcomes in the small intestine, evidenced by the elicitation of a pro-inflammatory response and decreased anti-inflammatory response in the duodenum, and by increased albumin transudation and decreased bifidobacterial growth in the distal region.

Aerobic training improves bone fragility by reducing the inflammatory microenvironment in bone tissue in type 2 diabetes.

Aerobic training (AT) is indicated in type 2 diabetes mellitus (T2DM) to control hyperglycaemia and inflammation. AT improves bone microarchitecture and resistance to fracture. The intensity of AT and the mechanisms that lead to the improvement in bone quality are still unknown. Using a mouse model of T2DM, we evaluated the effects of two intensities of forced AT. We divided mice into: sedentary (SED), T2DM-SED, low runners (LOW), T2DM-LOW, high runners (HIGH) and T2DM-HIGH. The AT for low was 8 m/minute (m/min); 5° slope or high 18 m/min; 15° slope for 2 months. We measured metabolic parameters, the serum cytokines concentration, lipocalin-2 (LCN-2) and adiponectin; and the tibial concentrations of LCN-2, tumour necrosis factor alpha (TNF-α) and protein carbonylation (CO). We evaluated femur morphometry and biomechanical properties. We performed multiple correlation analysis. The T2DM-LOW versus T2DM-SED group, shown an increase of interleukin (IL)-10 (417 ± 90 vs 102 ± 25 pg/mL) and improved trabecular bone (BV/TV: 31.8 ± 2.3 vs 19.25 ± 1.4%; Tb.Sp.: 1.62 ± 0.02 vs 2.0 ± 0.07 mm), by a decrease bone CO (3.4 ± 0.1 vs 6.0 ± 0.5 nmol/mg), bone TNF-α (84 ± 4 vs 239 ± 13 pg/mL) and LCN-2 (2887 ± 23 vs 3418 ± 105 pg/mL). The T2DM-HIGH versus T2DM-SED group showed a greater hypoglycaemic effect (228 ± 10 vs 408 ± 5 mg/dL), with improved cortical bone density (0.26 ± 0.012 vs 0.21 ± 0.007 mm) and fracture resistance (102 ± 8 vs 78 ± 5 MPa), by a reduction of bone TNF-α (77 ± 34 vs 239 ± 13 pg/mL); LCN-2 (2768 ± 20 vs 3418 ± 105 pg/mL) and CO (4.8 ± 0.5 vs 6.0 ± 0.5 nmol/mg). In conclusion, AT improves bone morphometry and biomechanical properties by reducing the bone inflammatory microenvironment.

Enforced mesenchymal stem cell tissue colonization counteracts immunopathology.

Mesenchymal stem/stromal cells (MSCs) are distributed within all tissues of the body. Though best known for generating connective tissue and bone, these cells also display immunoregulatory properties. A greater understanding of MSC cell biology is urgently needed because culture-expanded MSCs are increasingly being used in treatment of inflammatory conditions, especially life-threatening immune diseases. While studies in vitro provide abundant evidence of their immunomodulatory capacity, it is unknown whether tissue colonization of MSCs is critical to their ability to dampen/counteract evolving immunopathology in vivo. To address this question, we employed a murine model of fulminant immune-mediated inflammation, acute graft-versus-host disease (aGvHD), provoked by donor splenocyte-enriched full MHC-mismatched hematopoietic stem cell transplant. aGvHD induced the expression of E-selectin within lesional endothelial beds, and tissue-specific recruitment of systemically administered host-derived MSCs was achieved by enforced expression of HCELL, a CD44 glycoform that is a potent E-selectin ligand. Compared to mice receiving HCELL- MSCs, recipients of HCELL+ MSCs had increased MSC intercalation within aGvHD-affected site(s), decreased leukocyte infiltrates, lower systemic inflammatory cytokine levels, superior tissue preservation, and markedly improved survival. Mechanistic studies reveal that ligation of HCELL/CD44 on the MSC surface markedly potentiates MSC immunomodulatory activity by inducing MSC secretion of a variety of potent immunoregulatory molecules, including IL-10. These findings indicate that MSCs counteract immunopathology in situ, and highlight a role for CD44 engagement in unleashing MSC immunobiologic properties that maintain/establish tissue immunohomeostasis.

Moderate intensity aerobic training reduces the signs of peripheral sensitization in a mouse model of type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a global health problem for many reasons including the comorbidities, such as diabetic neuropathy (DPN), which is the most common. It has been suggested that aerobic training can improve metabolic health in individuals with T2DM. Still, the effect of aerobic training on DPN signs and its relationship with serum levels of tumor necrosis tumor alpha (TNF-α), an essential molecule in T2DM development, is unknown. We evaluated the effect of two intensities of aerobic training in adult male C57BL/6 mice divided into six groups: sedentary control (CTRL), control with low-intensity training (CTRL-LI), control with moderate-intensity training (CTRL-MI), T2DM sedentary (T2DM), T2DM with low-intensity training (T2DM-LI), and T2DM with moderate-intensity training (T2DM-MI). We induced the T2DM model by combining a hypercaloric diet and low doses of streptozotocin. We measured serum TNF-α levels and correlated them with peripheral sensitization and the cardinal signs of T2DM in mice. Moderate intensity aerobic training decreased the symptoms of DPN and improved metabolic health in T2DM. Interestingly, decreased TNF-α serum levels correlated with reduced peripheral thermal sensitivity and mechanical sensitivity by aerobic training. Moderate intensity aerobic training counteracts the development and symptoms of DPN and improve metabolic health in T2DM. Decreased TNF-α correlates with reduced peripheral thermal sensitivity and mechanical sensitivity by aerobic training.

Optimization of Mesenchymal Stromal Cell (MSC) Manufacturing Processes for a Better Therapeutic Outcome.

MSCs products as well as their derived extracellular vesicles, are currently being explored as advanced biologics in cell-based therapies with high expectations for their clinical use in the next few years. In recent years, various strategies designed for improving the therapeutic potential of mesenchymal stromal cells (MSCs), including pre-conditioning for enhanced cytokine production, improved cell homing and strengthening of immunomodulatory properties, have been developed but the manufacture and handling of these cells for their use as advanced therapy medicinal products (ATMPs) remains insufficiently studied, and available data are mainly related to non-industrial processes. In the present article, we will review this topic, analyzing current information on the specific regulations, the selection of living donors as well as MSCs from different sources (bone marrow, adipose tissue, umbilical cord, etc.), in-process quality controls for ensuring cell efficiency and safety during all stages of the manual and automatic (bioreactors) manufacturing process, including cryopreservation, the use of cell banks, handling medicines, transport systems of ATMPs, among other related aspects, according to European and US legislation. Our aim is to provide a guide for a better, homogeneous manufacturing of therapeutic cellular products with special reference to MSCs.

Moderate aerobic exercise on bone quality changes associated with aging and oxidative stress in BALB/c mice.

Exercise encourages active and healthy aging, maintaining functional and physical capabilities. This study aimed to assess the effects of a long-term moderate aerobic exercise protocol on bone microarchitecture and fragility associated with chronic inflammation and oxidative stress in aging. Male BALB/c mice (n = 10 per group) underwent a moderate exercise protocol from 13 weeks to 27 (adulthood age) or 108 weeks of age (elderly age) and were then sacrificed. Age-match sedentary mice were included as a control group. Serum cortisol concentrations were determined by chemiluminescent immunoassay, C-reactive protein (CRP) by a turbidimetric assay, advanced glycation end-products (AGEs) and malondialdehyde (MDA) by fluorescent spectroscopy, and total glutathione (GSH) by colorimetric method. The right femur was dissected formorphometric and densitometricanalysis bycomputerized microtomography (µCT),and biomechanical properties were assessed usinga three-point bending device. Musclefrom the same extremitywas obtained to determine relative mRNA expression ofpro-inflammatory cytokines (TNF-α and IL-6) by RT-qPCR.Statistical differences were evaluated by two-way ANOVA and Holm-Sidak method post hoc with P < 0.05. In elderly mice, moderate exercise increased glutathione levels and microarchitecture complexity but decreased bone fragility and oxidative stress markers, cortisol, and pro-inflammatory cytokines. In conclusion, these results suggest a strong link between a pro-inflammatory state and age-conditioned oxidative stress on bone quality. Thus, on a human scale, moderate aerobic exercise may improve bone quality during aging.

Docosahexaenoic acid improves altered mineralization proteins, the decreased quality of hydroxyapatite crystals and suppresses oxidative stress induced by high glucose.

Patients with type 2 diabetes mellitus (DM2) experience an increased risk of fractures and a variety of bone pathologies, such as osteoporosis. The suggested mechanisms of increased fracture risk in DM2 include chronic hyperglycaemia, which provokes oxidative stress, alters bone matrix, and decreases the quality of hydroxyapatite crystals. Docosahexaenoic acid (DHA), an omega-3 fatty acid, can increase bone formation, reduce bone loss, and it possesses antioxidant/anti-inflammatory properties. The present study aimed to determine the effect of DHA on altered osteoblast mineralisation and increased reactive oxygen species (ROS) induced by high glucose concentrations. A human osteoblast cell line was treated with 5.5 mM glucose (NG) or 24 mM glucose (HG), alone or in combination with 10 or 20 µM DHA. The collagen type 1 (Col1) scaffold, the expression of osteocalcin (OCN) and bone sialoprotein type-II (BSP-II), the alkaline phosphatase (ALP) specific activity, the mineral quality, the production of ROS and the mRNA expression of nuclear factor erythroid 2-related factor-2 (NRF2) were analysed. Osteoblasts cultured in HG and treated with either DHA concentration displayed an improved distribution of the Col1 scaffold, increased OCN and BSP-II expression, increased NRF2 mRNA, decreased ALP activity, carbonate substitution and reduced ROS production compared with osteoblasts cultured in HG alone. DHA counteracts the adverse effects of HG on bone mineral matrix quality and reduces oxidative stress, possibly by increasing the expression of NRF2.

Efficacy and safety of intramuscular administration of allogeneic adipose tissue derived and expanded mesenchymal stromal cells in diabetic patients with critical limb ischemia with no possibility of revascularization: study protocol for a randomized controlled double-blind phase II clinical trial (The NOMA Trial).

BACKGROUND: Chronic lower limb ischemia develops earlier and more frequently in patients with type 2 diabetes mellitus. Diabetes remains the main cause of lower-extremity non-traumatic amputations. Current medical treatment, based on antiplatelet therapy and statins, has demonstrated deficient improvement of the disease. In recent years, research has shown that it is possible to improve tissue perfusion through therapeutic angiogenesis. Both in animal models and humans, it has been shown that cell therapy can induce therapeutic angiogenesis, making mesenchymal stromal cell-based therapy one of the most promising therapeutic alternatives. The aim of this study is to evaluate the feasibility, safety, and efficacy of cell therapy based on mesenchymal stromal cells derived from adipose tissue intramuscular administration to patients with type 2 diabetes mellitus with critical limb ischemia and without possibility of revascularization. METHODS: A multicenter, randomized double-blind, placebo-controlled trial has been designed. Ninety eligible patients will be randomly assigned at a ratio 1:1:1 to one of the following: control group (n = 30), low-cell dose treatment group (n = 30), and high-cell dose treatment group (n = 30). Treatment will be administered in a single-dose way and patients will be followed for 12 months. Primary outcome (safety) will be evaluated by measuring the rate of adverse events within the study period. Secondary outcomes (efficacy) will be measured by assessing clinical, analytical, and imaging-test parameters. Tertiary outcome (quality of life) will be evaluated with SF-12 and VascuQol-6 scales. DISCUSSION: Chronic lower limb ischemia has limited therapeutic options and constitutes a public health problem in both developed and underdeveloped countries. Given that the current treatment is not established in daily clinical practice, it is essential to provide evidence-based data that allow taking a step forward in its clinical development. Also, the multidisciplinary coordination exercise needed to develop this clinical trial protocol will undoubtfully be useful to conduct academic clinical trials in the field of cell therapy in the near future. TRIAL REGISTRATION: ClinicalTrials.gov NCT04466007 . Registered on January 07, 2020. All items from the World Health Organization Trial Registration Data Set are included within the body of the protocol.

Use of cryopreserved human amniotic membrane in the treatment of skin ulcers secondary to calciphylaxis.

Calciphylaxis is a rare condition characterized by skin ulceration and necrosis as a result of vascular calcification of the small and medium blood vessels of skin and subcutaneous tissues. It mainly occurs in patients with advanced chronic kidney disease and sometimes leads to complications with a fatal outcome. In this report, we describe the case of a 67-year-old male patient with end stage renal disease presenting painful skin ulcers on his lower limbs. The lesions had progressively grown and were associated to severe pain and decreased quality of life. The ulcers did not respond to conventional treatments and the patient underwent skin biopsy of these lesions obtaining anatomopathological findings compatible with calciphylaxis. In this report, we present an innovative treatment for skin ulcers secondary to calciphylaxis using cryopreserved amniotic membrane (AM) as a dressing in order to promote epithelialization of the wounds. After four applications, healing of the main ulcer and reduction in pain was achieved. In summary, applying cryopreserved AM probed to be a promising strategy to reduce pain and to enhance epithelialization and healing of chronic non-responsive ulcers in calciphylaxis.

Exofucosylation of Adipose Mesenchymal Stromal Cells Alters Their Secretome Profile.

Mesenchymal stromal cells (MSCs) constitute the cell type more frequently used in many regenerative medicine approaches due to their exclusive immunomodulatory properties, and they have been reported to mediate profound immunomodulatory effects in vivo. Nevertheless, MSCs do not express essential adhesion molecules actively involved in cell migration, a phenotypic feature that hampers their ability to home inflamed tissues following intravenous administration. In this study, we investigated whether modification by fucosylation of murine AdMSCs (mAdMSCs) creates Hematopoietic Cell E-/L-selectin Ligand, the E-selectin-binding CD44 glycoform. This cell surface glycan modification of CD44 has previously shown in preclinical studies to favor trafficking of mAdMSCs to inflamed or injured peripheral tissues. We analyzed the impact that exofucosylation could have in other innate phenotypic and functional properties of MSCs. Compared to unmodified counterparts, fucosylated mAdMSCs demonstrated higher in vitro migration, an altered secretome pattern, including increased expression and secretion of anti-inflammatory molecules, and a higher capacity to inhibit mitogen-stimulated splenocyte proliferation under standard culture conditions. Together, these findings indicate that exofucosylation could represent a suitable cell engineering strategy, not only to facilitate the in vivo MSC colonization of damaged tissues after systemic administration, but also to convert MSCs in a more potent immunomodulatory/anti-inflammatory cell therapy-based product for the treatment of a variety of autoimmune, inflammatory, and degenerative diseases.

Bone Marrow-Derived Mononuclear Cell Transplants Decrease Retinal Gliosis in Two Animal Models of Inherited Photoreceptor Degeneration.

Inherited photoreceptor degenerations are not treatable diseases and a frequent cause of blindness in working ages. In this study we investigate the safety, integration and possible rescue effects of intravitreal and subretinal transplantation of adult human bone-marrow-derived mononuclear stem cells (hBM-MSCs) in two animal models of inherited photoreceptor degeneration, the P23H-1 and the Royal College of Surgeons (RCS) rat. Immunosuppression was started one day before the injection and continued through the study. The hBM-MSCs were injected in the left eyes and the animals were processed 7, 15, 30 or 60 days later. The retinas were cross-sectioned, and L- and S- cones, microglia, astrocytes and Müller cells were immunodetected. Transplantations had no local adverse effects and the CD45+ cells remained for up to 15 days forming clusters in the vitreous and/or a 2-3-cells-thick layer in the subretinal space after intravitreal or subretinal injections, respectively. We did not observe increased photoreceptor survival nor decreased microglial cell numbers in the injected left eyes. However, the injected eyes showed decreased GFAP immunoreactivity. We conclude that intravitreal or subretinal injection of hBM-MSCs in dystrophic P23H-1 and RCS rats causes a decrease in retinal gliosis but does not have photoreceptor neuroprotective effects, at least in the short term. However, this treatment may have a potential therapeutic effect that merits further investigation.

Sensory, Affective, and Cognitive Effects of Trigeminal Injury in Mice.

PURPOSE: To characterize adequate study of chronic neuropathic orofacial pain induced by a mental nerve injury in a mouse model, we propose a behavioral assessment of its dimensions: sensory, affective, and cognitive. MATERIALS AND METHODS: Trigeminal injury was induced by a chronic mental nerve constriction (MnC). Behavioral tests were conducted to assess the different dimensions of pain and to evaluate the general well-being of mice. RESULTS: Rodents who went through MnC showed signs of mechanical hyperalgesia and increased escape/avoidance behavior. They showed no alterations in general well-being behaviors, yet the injury was sufficient to induce impairment in the ability to adapt to the environmental requirements. CONCLUSIONS: MnC injury is an efficient model for the study of orofacial pain in mice, capable of inducing impairment in the different dimensions of pain. Intensity and temporality of its effects make our model less aggressive, yet effective to generate cognitive impairment. This work provides a solid foundation for the study of the neural circuits involved in the processing of neuropathic orofacial pain.

Intramuscular Injection of Bone Marrow Stem Cells in Amyotrophic Lateral Sclerosis Patients: A Randomized Clinical Trial.

BACKGROUND: Preclinical studies suggest that stem cells may be a valuable therapeutic tool in amyotrophic lateral sclerosis (ALS). As it has been demonstrated that there are molecular changes at the end-plate during the early stages of motorneuron degeneration in animal models, we hypothesize that the local effect of this stem cell delivery method could slow the progressive loss of motor units (MUs) in ALS patients. METHODS: We designed a Phase I/II clinical trial to study the safety of intramuscularly implanting autologous bone marrow mononuclear cells (BMMCs), including stem cells, in ALS patients and their possible effects on the MU of the tibialis anterior (TA) muscle. Twenty-two patients participated in a randomized, double-blind, placebo-controlled trial that consisted of a baseline visit followed by one intramuscular injection of BMNCs, follow-up visits at 30, 90, 180, and 360 days, and an additional year of clinical follow-up. In each patient, one TA muscle was injected with a single dose of BMMCs while the contralateral muscle was given a placebo; the sides were selected randomly. All visits included a complete EMG study of both TA muscles. RESULTS: Our results show that (1) the intramuscular injection of BMMCs is a safe procedure; (2) ALS patients show heterogeneities in the degree of TA injury; (3) a comparison of placebo-injected muscles with BMMC-injected muscles showed significant differences in only one parameter, the D50 index used to quantify the Compound Muscle Action Potential (CMAP) scan curve. This parameter was higher in the BMMC-injected TA muscle at both 90 days (placebo side: 29.55 ± 2.89, n = 20; experimental side: 39.25 ± 3.21, n = 20; p < 0.01) and 180 days (placebo side: 29.35 ± 3.29, n = 17; experimental side: 41.24 ± 3.34, n = 17; p < 0.01). CONCLUSION: This procedure had no effect on the TA muscle MU properties, with the exception of the D50 index. Finding differences in just this index supports the fact that it may be much more sensitive than other electrophysiological parameters when studying treatment effects. Given the low number of patients and their heterogeneity, these results justify exploring the efficacy of this procedure in further patients and other muscles, through Phase II trials. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov (identifier NCT02286011); EudraCT number 2011-004801-25.

Safety and Biodistribution of Human Bone Marrow-Derived Mesenchymal Stromal Cells Injected Intrathecally in Non-Obese Diabetic Severe Combined Immunodeficiency Mice: Preclinical Study.

Background: Mesenchymal stromal cells (MSCs) have potent immunomodulatory and neuroprotective properties, and have been tested in neurodegenerative diseases resulting in meaningful clinical improvements. Regulatory guidelines specify the need to perform preclinical studies prior any clinical trial, including biodistribution assays and tumourigenesis exclusion. We conducted a preclinical study of human bone marrow MSCs (hBM-MSCs) injected by intrathecal route in Non-Obese Diabetic Severe Combined Immunodeficiency mice, to explore cellular biodistribution and toxicity as a privileged administration method for cell therapy in Friedreich's Ataxia. Methods: For this purpose, 3 × 105 cells were injected by intrathecal route in 12 animals (experimental group) and the same volume of culture media in 6 animals (control group). Blood samples were collected at 24 h (n = 9) or 4 months (n = 9) to assess toxicity, and nine organs were harvested for histology and safety studies. Genomic DNA was isolated from all tissues, and mouse GAPDH and human ß2M and ß-actin genes were amplified by qPCR to analyze hBM-MSCs biodistribution. Results: There were no deaths nor acute or chronic toxicity. Hematology, biochemistry and body weight were in the range of normal values in all groups. At 24 h hBM-MSCs were detected in 4/6 spinal cords and 1/6 hearts, and at 4 months in 3/6 hearts and 1/6 brains of transplanted mice. No tumours were found. Conclusion: This study demonstrated that intrathecal injection of hBM-MSCs is safe, non toxic and do not produce tumors. These results provide further evidence that hBM-MSCs might be used in a clinical trial in patients with FRDA.