Treatment of medication-related osteonecrosis of the jaw with cell therapy.

Introduction: Medication-related osteonecrosis of the jaw (MRONJ) poses a significant challenge considering the absence of a "gold standard" treatment. Cell-based therapy and tissue engineering offer promising therapeutic alternatives. This study aimed to harness the regenerative properties of adipose-tissue stromal vascular fraction (AT-SVF) and leukocyte-platelet-rich fibrin (L-PRF) for MRONJ treatment. AT-SVF contains mesenchymal stromal cells (MSC) and endothelial progenitor cells (EPC), which promote bone formation, while the L-PRF scaffold can serve as a three-dimensional scaffold for the AT-SVF and support tissue healing through growth factor release. Materials and methods: The protocol involved applying autologous AT-SVF within an L-PRF matrix following surgical debridement. Age, gender, body mass index, comorbidities, underlying oncological condition, prescribed antiresorptive treatment: BP or DMB, antiresorptive treatment duration, antiresorptive treatment potential discontinuation, number of MRONJ lesion, MRONJ location, MRONJ stage, MRONJ trigger factor were assessed for each patient. Patients underwent the procedure and were monitored for a minimum of 6 months based on clinical, biological and medical imaging criteria. Results: Nine patients, with a total of ten MRONJ lesions, participated in the study. Six patients were female, and three were male, with a mean age of 68 ± 8 years. Four patients had multiple myeloma (MM), three had metastatic breast cancer, and two had metastatic prostate cancer. Seven MRONJ cases were classified as stage II, and three were classified as stage III. Soft tissue completely healed within a month after treatment in nine cases, with no clinical improvement observed in the remaining case. During follow-up, no sign of MRONJ recurrence was observed. Tridimensional medical imaging revealed bone healing 6 months after the surgical procedure. Immunophenotyping confirmed the presence of MSC and EPC in the AT-SVF: 12,6 ± 4,5% CD31+, 20.5 ± 7,8% CD34+, 34,4 ± 7,3% CD146+ and 54,6 ± 7,4% CD45+. Conclusion: This prospective study introduces a potential new treatment approach for MRONJ using autologous AT-SVF within an L-PRF scaffold. Our results are encouraging and suggest the need for further investigation with a larger patient cohort to better understand the underlying mechanisms.

Age-related changes in human bone marrow mesenchymal stromal cells: morphology, gene expression profile, immunomodulatory activity and miRNA expression.

Introduction: Mesenchymal stromal cells (MSC) are one of the main cellular components of bone marrow (BM) microenvironment. MSC play a key role in tissue regeneration, but they are also capable of immunomodulating activity. With host aging, MSC undergo age-related changes, which alter these functions, contributing to the set-up of "inflammaging", which is known to be the basis for the development of several diseases of the elderly, including cancer. However, there's few data investigating this facet of MSC, mainly obtained using murine models or replicative senescence. The aim of this research was to identify morphological, molecular and functional alterations of human bone marrow-derived MSC from young (yBM-MSC) and old (oBM-MSC) healthy donors. Methods: MSC were identified by analysis of cell-surface markers according to the ISCT criteria. To evaluate response to inflammatory status, MSC were incubated for 24h in the presence of IL-1ß, IFN-α, IFN-É£ and TNF-α. Macrophages were obtained by differentiation of THP-1 cells through PMA exposure. For M1 polarization experiments, a 24h incubation with LPS and IFN-É£ was performed. MSC were plated at the bottom of the co-culture transwell system for all the time of cytokine exposure. Gene expression was evaluated by real-time PCR after RNA extraction from BM-MSC or THP-1 culture. Secreted cytokines levels were quantitated through ELISA assays. Results: Aging MSC display changes in size, morphology and granularity. Higher levels of ß-Gal, reactive oxygen species (ROS), IL-6 and IL-8 and impaired colony-forming and cell cycle progression abilities were found in oBM-MSC. Gene expression profile seems to vary according to subjects' age and particularly in oBM-MSC seem to be characterized by an impaired immunomodulating activity, with a reduced inhibition of macrophage M1 status. The comparative analysis of microRNA (miRNA) expression in yBM-MSC and oBM-MSC revealed a significant difference for miRNA known to be involved in macrophage polarization and particularly miR-193b-3p expression is strongly increased after co-culture of macrophages with yBM-MSC. Conclusion: There are profound differences in terms of morphology, gene and miRNA expression and immunomodulating properties among yBM-MSC and oBM-MSC, supporting the critical role of aging BM microenvironment on senescence, immune-mediated disorders and cancer pathogenesis.

Extracellular vesicles in hematological malignancies: EV-dence for reshaping the tumoral microenvironment.

Following their discovery at the end of the 20th century, extracellular vesicles (EVs) ranging from 50-1,000 nm have proven to be paramount in the progression of many cancers, including hematological malignancies. EVs are a heterogeneous group of cell-derived membranous structures that include small EVs (commonly called exosomes) and large EVs (microparticles). They have been demonstrated to participate in multiple physiological and pathological processes by allowing exchange of biological material (including among others proteins, DNA and RNA) between cells. They are therefore a crucial way of intercellular communication. In this context, malignant cells can release these extracellular vesicles that can influence their microenvironment, induce the formation of a tumorigenic niche, and prepare and establish distant niches facilitating metastasis by significantly impacting the phenotypes of surrounding cells and turning them toward supportive roles. In addition, EVs are also able to manipulate the immune response and to establish an immunosuppressive microenvironment. This in turn allows for ideal conditions for heightened chemoresistance and increased disease burden. Here, we review the latest findings and reports studying the effects and therapeutic potential of extracellular vesicles in various hematological malignancies. The study of extracellular vesicles remains in its infancy; however, rapid advances in the analysis of these vesicles in the context of disease allow us to envision prospects to improve the detection and treatment of hematological malignancies.

Acute myeloid leukemia-derived exosomes deliver miR-24-3p to hinder the T-cell immune response through DENN/MADD targeting in the NF-κB signaling pathways.

BACKGROUND: microRNAs (miRNAs) are known as potent gene expression regulators, and several studies have revealed the prognostic value of miRNAs in acute myeloid leukemia (AML) patient survival. Recently, strong evidence has indicated that miRNAs can be transported by exosomes (EXOs) from cancer cells to recipient immune microenvironment (IME) cells. RESULTS: We found that AML blast-released EXOs enhance CD3 T-cell apoptosis in both CD4 and CD8 T cells. We hypothesized that miRNAs present in EXOs are key players in mediating the changes observed in AML T-cell survival. We found that miR-24-3p, a commonly overexpressed miRNA in AML, was present in released EXOs, suggesting that EXO-miR-24-3p was linked to the increased miR-24-3p levels detected in isolated AML T cells. These results were corroborated by ex vivo-generated miR-24-3p-enriched EXOs, which showed that miR-24-3p-EXOs increased apoptosis and miR-24-3p levels in T cells. We also demonstrated that overexpression of miR-24-3p increased T-cell apoptosis and affected T-cell proliferation by directly targeting DENN/MADD expression and indirectly altering the NF-κB, p-JAK/STAT, and p-ERK signaling pathways but promoting regulatory T-cell (Treg) development. CONCLUSIONS: These results highlight a mechanism through which AML blasts indirectly impede T-cell function via transferred exosomal miR-24-3p. In conclusion, by characterizing the signaling network regulated by individual miRNAs in the leukemic IME, we aimed to discover new nonleukemic immune targets to rescue the potent antitumor function of T cells against AML blasts. Video Abstract.

Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stromal Cells as an Efficient Nanocarrier to Deliver siRNA or Drug to Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Treatment of PDAC remains a major challenge. This study aims to evaluate, in vitro, the use of human umbilical cord mesenchymal stromal cell (UC-MSC)-derived EVs to specifically target pancreatic cancer cells. EVs were isolated from the FBS-free supernatants of the cultured UC-MSCs by ultracentrifugation and characterized by several methods. EVs were loaded with scramble or KRASG12D-targeting siRNA by electroporation. The effects of control and loaded EVs on different cell types were evaluated by assessing cell proliferation, viability, apoptosis and migration. Later, the ability of EVs to function as a drug delivery system for doxorubicin (DOXO), a chemotherapeutic drug, was also evaluated. Loaded EVs exhibited different kinetic rates of uptake by three cell lines, namely, BxPC-3 cells (pancreatic cancer cell line expressing KRASwt), LS180 cells (colorectal cell line expressing KRASG12D) and PANC-1 cells (pancreatic cell line expressing KRASG12D). A significant decrease in the relative expression of the KRASG12D gene after incubation with KRAS siRNA EVs was observed by real-time PCR. KRASG12D siRNA EVs significantly reduced the proliferation, viability and migration of the KRASG12D cell lines compared to scramble siRNA EVs. An endogenous EV production method was applied to obtain DOXO-loaded EVs. Briefly, UC-MSCs were treated with DOXO. After 24 h, UC-MSCs released DOXO-loaded EVs. DOXO-loaded EVs were rapidly taken up by PANC-1 cells and induced apoptotic cell death more efficiently than free DOXO. In conclusion, the use of UC-MSC-derived EVs as a drug delivery system for siRNAs or drugs could be a promising approach for the targeted treatment of PDAC.

Potential of Mesenchymal Stromal Cell-Derived Extracellular Vesicles as Natural Nanocarriers: Concise Review.

Intercellular communication, through direct and indirect cell contact, is mandatory in multicellular organisms. These last years, the microenvironment, and in particular, transfer by extracellular vesicles (EVs), has emerged as a new communication mechanism. Different biological fluids and cell types are common sources of EVs. EVs play different roles, acting as signalosomes, biomarkers, and therapeutic agents. As therapeutic agents, MSC-derived EVs display numerous advantages: they are biocompatible, non-immunogenic, and stable in circulation, and they are able to cross biological barriers. Furthermore, EVs have a great potential for drug delivery. Different EV isolation protocols and loading methods have been tested and compared. Published and ongoing clinical trials, and numerous preclinical studies indicate that EVs are safe and well tolerated. Moreover, the latest studies suggest their applications as nanocarriers. The current review will describe the potential for MSC-derived EVs as drug delivery systems (DDS) in disease treatment, and their advantages. Thereafter, we will outline the different EV isolation methods and loading techniques, and analyze relevant preclinical studies. Finally, we will describe ongoing and published clinical studies. These elements will outline the benefits of MSC-derived EV DDS over several aspects.

Suburethral implantation of autologous regenerative cells for female stress urinary incontinence management: Results of a pilot study.

OBJECTIVES: To assess the feasibility and the safety of treating female stress urinary incontinence (SUI) with suburethral implantation of a mixture of the stromal vascular fraction from adipose tissue and leukocyte-and platelet-rich-fibrin. METHODS: Patients with SUI were treated with a mixture of stromal vascular fraction and leukocyte-and platelet-rich fibrin. The stromal vascular fraction was obtained from enzymatic digestion of autologous adipose-tissue and added to an leukocyte-and platelet-rich-fibrin membrane. The mixture was transvaginally implanted into the suburethral area. A fraction of the Stromal vascular fraction sample was used for cellular characterization. Patients were followed for 9 months. Every 3 months, the patients were clinically evaluated with a cough- stress test and a validated-questionnaire. An MRI was performed preoperatively and 3 months after the procedure to assess tissue changes. RESULTS: Ten patients received the surgical procedure. The validated-questionnaire revealed a subjective SUI improvement in nine patients 3 months after the operation and in seven patients 9 months after the operation. Eight, six, and four patients achieved a negative cough-stress test 3, 6 and 9 months post-injection, respectively. Flow cytometric analysis of stromal vascular fraction cell phenotypes revealed predominantly mesenchymal and endothelial cell heterogeneity. In total, we injected 0,18 × 106 to 13,6 × 106 cells. No adverse events were observed peri- or postoperatively. CONCLUSION: These preliminary results suggest that the suburethral implantation of a combination of SVF and l-PRF is a feasible and safe modality for treating female SUI. However, evidence is lacking and further research are needed to clarify the respective roles of SVF and l-PRF in female SUI treatment.

Bioscreening and pre-clinical evaluation of the impact of bioactive molecules from Ptychotis verticillata on the multilineage potential of mesenchymal stromal cells towards immune- and inflammation-mediated diseases.

OBJECTIVE AND DESIGN: Mesenchymal stromal cells (MSCs) are currently used in cell reparative medicine due to their trophic and ant-inflammatory properties. The modulation of stem cell properties by phytochemicals has been suggested as a tool to empower their tissue repair capacity. In vitro, MSCs are characterized by their tri-lineage potential that holds great interest for tissue regeneration. Ptychotis Verticillata (PV), an aromatic and medicinal plant, may be thus used to modulate the in vitro multilineage potential of MSCs. MATERIALS AND METHODS: We screened the impact of PV-derived essential oil and their bioactive molecules (thymol and carvacrol) on the in vitro multilineage potential of MSCs. Different concentrations and incubation times of these compounds were assessed during the osteogenesis and adipogenesis of MSCs. RESULTS: The analysis of 75 conditions indicates that these compounds are biologically active by promoting two major differentiation lineages from MSCs. In a time- and dose-dependent manner, thymol and carvacrol increased the osteogenesis and adipogenesis. CONCLUSION: According to these preliminary observations, the addition of PV extract may stimulate the tissue regenerative and repair functions of MSCs. Further optimization of compound extraction and characterization from PV as well as cell treatment conditions should increase their therapeutic value in combination with MSCs.

In Vitro Cellular and Molecular Interplay between Human Foreskin-Derived Mesenchymal Stromal/Stem Cells and the Th17 Cell Pathway.

Foreskin, considered a biological waste material, has been shown to be a reservoir of therapeutic cells. The immunomodulatory properties of mesenchymal stromal/stem cells (MSCs) from the foreskin (FSK-MSCs) are being evaluated in cell-based therapy for degenerative, inflammatory and autoimmune disorders. Within the injured/inflamed tissue, proinflammatory lymphocytes such as IL-17-producing T helper cells (Th17) may interact with the stromal microenvironment, including MSCs. In this context, MSCs may encounter different levels of T cells as well as specific inflammatory signals. Uncovering the cellular and molecular changes during this interplay is central for developing an efficient and safe immunotherapeutic tool. To this end, an in vitro human model of cocultures of FSK-MSCs and T cells was established. These cocultures were performed at different cell ratios in the presence of an inflammatory setting. After confirming that FSK-MSCs respond to ISCT criteria by showing a typical phenotype and multilineage potential, we evaluated by flow cytometry the expression of Th17 cell markers IL-17A, IL23 receptor and RORγt within the lymphocyte population. We also measured 15 human Th17 pathway-related cytokines. Regardless of the T cell/MSC ratio, we observed a significant increase in IL-17A expression associated with an increase in IL-23 receptor expression. Furthermore, we observed substantial modulation of IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α secretion. These findings suggest that FSK-MSCs are receptive to their environment and modulate the T cell response accordingly. The changes within the secretome of the stromal and immune environment are likely relevant for the therapeutic effect of MSCs. FSK-MSCs represent a valuable cellular product for immunotherapeutic purposes that needs to be further clarified and developed.

The Therapeutic Potential of Mesenchymal Stromal Cells for Regenerative Medicine: Current Knowledge and Future Understandings.

In recent decades, research on the therapeutic potential of progenitor cells has advanced considerably. Among progenitor cells, mesenchymal stromal cells (MSCs) have attracted significant interest and have proven to be a promising tool for regenerative medicine. MSCs are isolated from various anatomical sites, including bone marrow, adipose tissue, and umbilical cord. Advances in separation, culture, and expansion techniques for MSCs have enabled their large-scale therapeutic application. This progress accompanied by the rapid improvement of transplantation practices has enhanced the utilization of MSCs in regenerative medicine. During tissue healing, MSCs may exhibit several therapeutic functions to support the repair and regeneration of injured tissue. The process underlying these effects likely involves the migration and homing of MSCs, as well as their immunotropic functions. The direct differentiation of MSCs as a cell replacement therapeutic mechanism is discussed. The fate and behavior of MSCs are further regulated by their microenvironment, which may consequently influence their repair potential. A paracrine pathway based on the release of different messengers, including regulatory factors, chemokines, cytokines, growth factors, and nucleic acids that can be secreted or packaged into extracellular vesicles, is also implicated in the therapeutic properties of MSCs. In this review, we will discuss relevant outcomes regarding the properties and roles of MSCs during tissue repair and regeneration. We will critically examine the influence of the local microenvironment, especially immunological and inflammatory signals, as well as the mechanisms underlying these therapeutic effects. Importantly, we will describe the interactions of local progenitor and immune cells with MSCs and their modulation during tissue injury. We will also highlight the crucial role of paracrine pathways, including the role of extracellular vesicles, in this healing process. Moreover, we will discuss the therapeutic potential of MSCs and MSC-derived extracellular vesicles in the treatment of COVID-19 (coronavirus disease 2019) patients. Overall, this review will provide a better understanding of MSC-based therapies as a novel immunoregenerative strategy.

Transcriptional Profile of Cytokines, Regulatory Mediators and TLR in Mesenchymal Stromal Cells after Inflammatory Signaling and Cell-Passaging.

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1ß, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFß. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches.

Cross-Talk Between Mesenchymal Stromal Cells (MSCs) and Endothelial Progenitor Cells (EPCs) in Bone Regeneration.

Bone regeneration is a complex, well-orchestrated process based on the interactions between osteogenesis and angiogenesis, observed in both physiological and pathological situations. However, specific conditions (e.g., bone regeneration in large quantity, immunocompromised regenerative process) require additional support. Tissue engineering offers novel strategies. Bone regeneration requires a cell source, a matrix, growth factors and mechanical stimulation. Regenerative cells, endowed with proliferation and differentiation capacities, aim to recover, maintain, and improve bone functions. Vascularization is mandatory for bone formation, skeletal development, and different osseointegration processes. The latter delivers nutrients, growth factors, oxygen, minerals, etc. The development of mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs) cocultures has shown synergy between the two cell populations. The phenomena of osteogenesis and angiogenesis are intimately intertwined. Thus, cells of the endothelial line indirectly foster osteogenesis, and conversely, MSCs promote angiogenesis through different interaction mechanisms. In addition, various studies have highlighted the importance of the microenvironment via the release of extracellular vesicles (EVs). These EVs stimulate bone regeneration and angiogenesis. In this review, we describe (1) the phenomenon of bone regeneration by different sources of MSCs. We assess (2) the input of EPCs in coculture in bone regeneration and describe their contribution to the osteogenic potential of MSCs. We discuss (3) the interaction mechanisms between MSCs and EPCs in the context of osteogenesis: direct or indirect contact, production of growth factors, and the importance of the microenvironment via the release of EVs.

Mesenchymal Stem/Stromal Cell Therapeutic Features: The Bridge between the Bench and the Clinic.

Mesenchymal stem/stromal cells (MSCs) are considered a relevant therapeutic product for various clinical applications [...].

Priming of mesenchymal stem cells with a hydrosoluble form of curcumin allows keeping their mesenchymal properties for cell-based therapy development.

Mesenchymal stem cells are increasingly studied for their use as drug-carrier in addition to their intrinsic potential for regenerative medicine. They could be used to transport molecules with a poor bioavailability such as curcumin in order to improve their clinical usage. This natural polyphenol, well-known for its antioxidant and anti-inflammatory properties, has a poor solubility that limits its clinical potential. For this purpose, the use of NDS27, a curcumin salt complexed with hydroxypropyl-beta-cyclodextrin (HPßCD), displaying an increased solubility in aqueous solution, is preferred. This study aims to evaluate the uptake of NDS27 into skeletal muscle-derived mesenchymal stem cells (mdMSCs) and the effects of such uptake onto their mesenchymal properties. It appeared that the uptake of NDS27 into mdMSCs is concentration-dependent and not time-dependent. The use of a concentration of 7 µmol/L which does not affect the viability and proliferation also allows preservation of their adhesion, invasion and T cell immunomodulatory abilities.

New Anti-Leukemic Effect of Carvacrol and Thymol Combination through Synergistic Induction of Different Cell Death Pathways.

Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells, and treatment for AML is lengthy and can be very expensive. Medicinal plants and their bioactive molecules are potential candidates for improving human health. In this work, we studied the effect of Ptychotis verticillata (PV) essential oil and its derivatives, carvacrol and thymol, in AML cell lines. We demonstrated that a combination of carvacrol and thymol induced tumor cell death with low toxicity on normal cells. Mechanistically, we highlighted that different molecular pathways, including apoptosis, oxidative, reticular stress, autophagy, and necrosis, are implicated in this potential synergistic effect. Using quantitative RT-PCR, Western blotting, and apoptosis inhibitors, we showed that cell death induced by the carvacrol and thymol combination is caspase-dependent in the HL60 cell line and caspase-independent in the other cell lines tested. Further investigations should focus on improving the manufacturing of these compounds and understanding their anti-tumoral mechanisms of action. These efforts will lead to an increase in the efficiency of the oncotherapy strategy regarding AML.

Immuno-comparative screening of adult-derived human liver stem/progenitor cells for immune-inflammatory-associated molecules.

OBJECTIVE: One of the main challenges in liver cell therapy is the replacement of damaged cells and the induction of a tolerogenic microenvironment to promote graft acceptance by the recipient. Adult-derived human liver stem/progenitor cells (ADHLSCs) are currently evaluated at the clinical levels as a promising pro-regenerative and immune-modulatory tool. The expression profile of several immunological molecules may influence the local immune-inflammatory response and, therefore, modulate the tissue healing process. To increase the quality and safety of ADHLSCs before transplantation requires an appropriate analysis and characterization of their pattern expression of immune-inflammatory-associated molecules. METHODS: The expression of 27 molecules belonging to T-cell co-stimulatory pathway, CD47 partners, Ikaros family, CD300 family and TNF family were analyzed using flow cytometry. We compared their expression profiles to PBMCs, hepatocytes and ADHLSCs in both expansion and after hepatogenic differentiation culture conditions. RESULTS: This original immuno-comparative screening revealed that liver cell populations do not constitutively present significant immunological pattern compared to PBMCs. Moreover, our findings highlight that neither the expansion nor the hepatogenic differentiation induces the expression of immune-inflammatory molecules. The detailed expression characteristics (percentage of positive cells and median fluorescence intensity) of each molecule were analyzed and presented. CONCLUSION: By analyzing 27 relevant molecules, our immuno-comparative screening demonstrates that ADHLSCs keep a non-immunogenic profile independent of their expansion or hepatogenic differentiation state. Accordingly, the immunological profile of ADHLSCs seems to support their safe and efficient use in liver tissue therapeutic repair strategy.

Therapeutic Mesenchymal Stem/Stromal Cells: Value, Challenges and Optimization.

Cellular therapy aims to replace damaged resident cells by restoring cellular and molecular environments suitable for tissue repair and regeneration. Among several candidates, mesenchymal stem/stromal cells (MSCs) represent a critical component of stromal niches known to be involved in tissue homeostasis. In vitro, MSCs appear as fibroblast-like plastic adherent cells regardless of the tissue source. The therapeutic value of MSCs is being explored in several conditions, including immunological, inflammatory and degenerative diseases, as well as cancer. An improved understanding of their origin and function would facilitate their clinical use. The stemness of MSCs is still debated and requires further study. Several terms have been used to designate MSCs, although consensual nomenclature has yet to be determined. The presence of distinct markers may facilitate the identification and isolation of specific subpopulations of MSCs. Regarding their therapeutic properties, the mechanisms underlying their immune and trophic effects imply the secretion of various mediators rather than direct cellular contact. These mediators can be packaged in extracellular vesicles, thus paving the way to exploit therapeutic cell-free products derived from MSCs. Of importance, the function of MSCs and their secretome are significantly sensitive to their environment. Several features, such as culture conditions, delivery method, therapeutic dose and the immunobiology of MSCs, may influence their clinical outcomes. In this review, we will summarize recent findings related to MSC properties. We will also discuss the main preclinical and clinical challenges that may influence the therapeutic value of MSCs and discuss some optimization strategies.

Importance of Crosstalk Between Chronic Lymphocytic Leukemia Cells and the Stromal Microenvironment: Direct Contact, Soluble Factors, and Extracellular Vesicles.

Chronic lymphocytic leukemia (CLL) is caused by the accumulation of malignant B cells due to a defect in apoptosis and the presence of small population of proliferating cells principally in the lymph nodes. The abnormal survival of CLL B cells is explained by a plethora of supportive stimuli produced by the surrounding cells of the microenvironment, including follicular dendritic cells (FDCs), and mesenchymal stromal cells (MSCs). This crosstalk between malignant cells and normal cells can take place directly by cell-to-cell contact (assisted by adhesion molecules such as VLA-4 or CD100), indirectly by soluble factors (chemokines such as CXCL12, CXCL13, or CCL2) interacting with their receptors or by the exchange of material (protein, microRNAs or long non-coding RNAs) via extracellular vesicles. These different communication methods lead to different activation pathways (including BCR and NFκB pathways), gene expression modifications (chemokines, antiapoptotic protein increase, prognostic biomarkers), chemotaxis, homing in lymphoid tissues and survival of leukemic cells. In addition, these interactions are bidirectional, and CLL cells can manipulate the normal surrounding stromal cells in different ways to establish a supportive microenvironment. Here, we review this complex crosstalk between CLL cells and stromal cells, focusing on the different types of interactions, activated pathways, treatment strategies to disrupt this bidirectional communication, and the prognostic impact of these induced modifications.

Impact of Bone Marrow miR-21 Expression on Acute Myeloid Leukemia T Lymphocyte Fragility and Dysfunction.

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy in which antitumor immunity is impaired. The therapeutic management of AML requires understanding the mechanisms involved in the fragility and immune dysfunction of AML T lymphocytes. METHODS: In this study, T lymphocytes from healthy donors (HD) and AML patients were used. Extracellular vesicles (EVs) from leukemic cells were screened for their microRNA content and impact on T lymphocytes. Flow cytometry, transcriptomic as well as lentiviral transduction techniques were used to carry out the research. RESULTS: We observed increased cell death of T lymphocytes from AML patients. EVs from leukemia myeloid cell lines harbored several miRNAs, including miR-21, and were able to induce T lymphocyte death. Compared to that in HD, miR-21 was overexpressed in both the bone marrow fluid and infiltrating T lymphocytes of AML patients. MiR-21 induces T lymphocyte cell death by upregulating proapoptotic gene expression. It also increases the immunosuppressive profile of T lymphocytes by upregulating the IL13, IL4, IL10, and FoxP3 genes. CONCLUSIONS: Our results demonstrate that miR-21 plays a significant role in AML T lymphocyte dysfunction and apoptosis. Targeting miR-21 may be a novel approach to restore the efficacy of the immune response against AML.

Inflammation Differentially Modulates the Biological Features of Adult Derived Human Liver Stem/Progenitor Cells.

The progression of mesenchymal stem cell-based therapy from concept to cure closely depends on the optimization of conditions that allow a better survival and favor the cells to achieve efficient liver regeneration. We have previously demonstrated that adult-derived human liver stem/progenitor cells (ADHLSC) display significant features that support their clinical development. The current work aims at studying the impact of a sustained pro-inflammatory environment on the principal biological features of ADHLSC in vitro. METHODS: ADHLSC from passages 4-7 were exposed to a cocktail of inflammatory cytokines for 24 h and 9 days and subsequently analyzed for their viability, expression, and secretion profiles by using flow cytometry, RT-qPCR, and antibody array assay. The impact of inflammation on the hepatocytic differentiation potential of ADHLSC was also evaluated. RESULTS: ADHLSC treated with a pro-inflammatory cocktail displayed significant decrease of cell yield at both times of treatment while cell mortality was observed at 9 days post-priming. After 24 h, no significant changes in the immuno-phenotype of ADHLSC expression profile could be noticed while after 9 days, the expression profile of relevant markers has changed both in the basal conditions and after inflammation treatment. Inflammation cocktail enhanced the release of IL-6, IL-8, CCL5, monocyte-chemo-attractant protein-2 and 3, CXCL1/GRO, and CXCL5/ENA78. Furthermore, while IP-10 secretion was increased after 24 h priming, granulocyte macrophage colony-stimulating factor enhanced secretion was noticed after 9 days treatment. Finally, priming of ADHLSC did not affect their potential to differentiate into hepatocyte-like cells. CONCLUSION: These results indicate that ADHLSCs are highly sensitive to inflammation and respond to such signals by adjusting their gene and protein expression. Accordingly, monitoring the inflammatory status of patients at the time of cell transplantation, will certainly help in enhancing ADHLSC safety and efficiency.