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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.

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.

β-thalassemia is a disease caused by genetic mutations including a nucleotide change, small insertions or deletions in the β-globin gene, or in rare cases, gross deletions into the β-globin gene. These mutations affect globin-chain subunits within the hemoglobin tetramer what induces an imbalance in the α/β-globin chain ratio, with an excess of free α-globin chains that triggers the most important pathogenic events of the disease: ineffective erythropoiesis, chronic anemia/chronic hypoxia, compensatory hemopoietic expansion and iron overload. Based on advances in our knowledge of the pathophysiology of β-thalassemia, in recent years, emerging therapies and clinical trials are being conducted and are classified into three major categories based on the different approach features of the underlying pathophysiology: correction of the α/β-globin disregulation; improving iron overload and reverse ineffective erythropoiesis. However, pathways such as the dysregulation of transcriptional factors, activation of the inflammasome, or approach to mechanisms of bone mineral loss, remain unexplored for future therapeutic targets. In this review, we update the main pathophysiological pathways involved in β-thalassemia, focusing on the development of new therapies directed at new therapeutic targets.

Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.

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.

Background: The lack of knowledge of the progression mechanisms of glioblastoma (GB), the most aggressive brain tumor, contributes to the absence of successful therapeutic strategies. Our team has recently demonstrated a crucial new role for chaperone-mediated autophagy (CMA) in pericytes (PC)-acquired immunosuppressive function, which prevents anti-tumor immune responses and facilitates GB progression. The possible impact that GB-induced CMA in PC has on other functions that might be useful for future GB prognosis/treatment, has not been explored yet. Thus, we proposed to analyze the contribution of CMA to other GB-induced changes in PC biology and determine if CMA ablation in PC is a key target mechanism for GB treatment.Methods: Studies of RNA-seq and secretome analysis were done in GB-conditioned PC with and without CMA (from knockout mice for LAMP-2A) and compared to control PC. Different therapeutic strategies in a GB mouse model were compared.Results: We found several gene expression pathways enriched in LAMP2A-KO PC and affected by GB-induced CMA in PC that correlate with our previous findings. Phagosome formation, cellular senescence, focal adhesion and the effector function to promote anti-tumor immune responses were the most affected pathways, revealing a transcriptomic profiling of specific target functions useful for future therapies. In addition, several molecules associated with tumor mechanisms and related to tumor immune responses such as gelsolin, periostin, osteopontin, lumican and vitamin D, were identified in the PC secretome dependent on GB-induced CMA. The CMA ablation in PC with GB cells showed an expected immunogenic phenotype able to phagocyte GB cells and a key strategy to develop future therapeutic strategies against GB tumor progression. A novel intravenous therapy using exofucosylated CMA-deficient PC was efficient to make PC reach the tumor niche and facilitate tumor elimination.Conclusion: Our results corroborate previous findings on the impaired immunogenic function of PC with GB-induced CMA, driving to other altered PC functions and the identifications of new target markers related to the tumor immune responses and useful for GB prognosis/therapy. Our work demonstrates CMA ablation in PC as a key target mechanism to develop a successful therapy against GB progression.

BackgroundPreclinical 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.MethodsWe 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.ResultsOur 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).ConclusionThis 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 Registrationwww.clinicaltrials.gov (identifier NCT02286011); EudraCT number 2011-004801-25.

Cell therapy is a progressively growing field that is rapidly moving from preclinical model development to clinical application. Outcomes obtained from clinical trials reveal the therapeutic potential of stem cell-based therapy to deal with unmet medical treatment needs for several disorders with no therapeutic options. Among adult stem cells, mesenchymal stem cells (MSCs) are the leading cell type used in advanced therapies for the treatment of autoimmune, inflammatory and vascular diseases. To date, the safety and feasibility of autologous MSC-based therapy has been established; however, their indiscriminate use has resulted in mixed outcomes in preclinical and clinical studies. While MSCs derived from diverse tissues share common properties depending on the type of clinical application, they markedly differ within clinical trials in terms of efficacy, resulting in many unanswered questions regarding the application of MSCs. Additionally, our experience in clinical trials related to critical limb ischemia pathology (CLI) shows that the therapeutic efficacy of these cells in different animal models has only been partially reproduced in humans through clinical trials. Therefore, it is crucial to develop new research to identify pitfalls, to optimize procedures and to clarify the repair mechanisms used by these cells, as well as to be able to offer a next generation of stem cell that can be routinely used in a cost-effective and safe manner in stem cell-based therapies targeting CLI.