K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

Adenoviral vectors are important vehicles for delivering therapeutic genes into mammalian cells. However, the yield of the adenoviral transduction of murine mesenchymal stromal cells (MSC) is low. Here, we aimed to improve the adenoviral transduction efficiency of bone marrow-derived MSC. Our data showed that among all the potential transduction boosters that we tested, the K2 Transfection System (K2TS) greatly increased the transduction efficiency. After optimization of both K2TS components, the yield of the adenoviral transduction increased from 18% to 96% for non-obese diabetic (NOD)-derived MSC, from 30% to 86% for C57BL/6-derived MSC, and from 0.6% to 63% for BALB/c-derived MSC, when 250 transduction units/cell were used. We found that MSC derived from these mouse strains expressed different levels of the coxsackievirus and adenovirus receptors (MSC from C57BL/6≥NOD>>>BALB/c). K2TS did not increase the level of the receptor expression, but desensitized the cells to foreign DNA and facilitated the virus entry into the cell. The expression of Stem cells antigen-1 (Sca-1) and 5′-nucleotidase (CD73) MSC markers, the adipogenic and osteogenic differentiation potential, and the immunosuppressive capacity were preserved after the adenoviral transduction of MSC in the presence of the K2TS. In conclusion, K2TS significantly enhanced the adenoviral transduction of MSC, without interfering with their main characteristics and properties.     

HATMSC Secreted Factors in the Hydrogel as a Potential Treatment for Chronic Wounds—In Vitro Study

Mesenchymal stem cells (MSCs) can improve chronic wound healing; however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.     

Mesenchymal Stem Cells Can Both Enhance and Inhibit the Cellular Response to DNA Immunization by Genes of Nonstructural Proteins of the Hepatitis C Virus

Despite extensive research, there is still no vaccine against the hepatitis C virus (HCV). The aim of this study was to investigate whether MSCs can exhibit adjuvant properties during DNA vaccination against hepatitis C. We used the pcNS3-NS5B plasmid encoding five nonstructural HCV proteins and MSCs derived from mice bone marrow. Five groups of DBA mice were immunized with the plasmid and/or MSCs in a different order. Group 1 was injected with the plasmid twice at intervals of 3 weeks; Group 2 with the plasmid, and after 24 h with MSCs; Group 3 with MSCs followed by the plasmid the next day; Group 4 with only MSCs; and Group 5 with saline. When the MSCs were injected prior to DNA immunization, the cell immune response to HCV proteins assessed by the level of IFN-γ synthesis was markedly increased compared to DNA alone. In contrast, MSCs injected after DNA suppressed the immune response. Apparently, the high level of proinflammatory cytokines detected after DNA injection promotes the conversion of MSCs introduced later into the immunosuppressive MSC2. The low level of cytokines in mice before MSC administration promotes the high immunostimulatory activity of MSC1 in response to a DNA vaccine. Thus, when administered before DNA, MSCs are capable of exhibiting promising adjuvant properties.     

Characteristics of MSCs in Synovial Fluid and Mode of Action of Intra-Articular Injections of Synovial MSCs in Knee Osteoarthritis

We have been studying mesenchymal stem cells (MSCs) in synovial fluid and the intra-articular injection of synovial MSCs in osteoarthritis (OA) knees. Here, mainly based on our own findings, we overview the characteristics of endogenous MSCs in the synovial fluid of OA knees and their mode of action when injected exogenously into OA knees. Many MSCs similar to synovial MSCs were detected in the synovial fluid of human OA knees, and their number correlated with the radiological OA grade. Our suspended synovium culture model demonstrated the release of MSCs from the synovium through a medium into a non-contacting culture dish. In OA knees, endogenous MSCs possibly mobilize in a similar manner from the synovium through the synovial fluid and act protectively. However, the number of mobilized MSCs is limited; therefore, OA progresses in its natural course. Synovial MSC injections inhibited the progression of cartilage degeneration in a rat OA model. Injected synovial MSCs migrated into the synovium, maintained their MSC properties, and increased the gene expressions of TSG-6, PRG-4, and BMP-2. Exogenous synovial MSCs can promote anti-inflammation, lubrication, and cartilage matrix synthesis in OA knees. Based on our findings, we have initiated a human clinical study of synovial MSC injections in OA knees.     

Hematopoietic–Mesenchymal Signals Regulate the Properties of Mesenchymal Stem Cells

It is well known that the properties of hematopoietic stem/progenitor cells (HSCs), such as their self-renewal ability and multipotency, are maintained through interactions with mesenchymal stem/stromal cells (MSCs). MSCs are rare cells that are present in the bone marrow and are useful for clinical applications due to their functional ability. To obtain the necessary number of cells, MSCs must be cultured to expand, but this causes a remarkable decrease in stem cell properties, such as multipotency and proliferation ability. In this study, we show that the c-Mpl signal, which is related to the maintenance of hematopoietic stem cells, has an important effect on the proliferation and differentiation ability of MSCs. Utilizing a co-culture system comprising MSCs and HSCs, it is suggested that signaling from hematopoietic cells to MSCs supports cell proliferation. Interestingly, the enhanced proliferation ability of the HSCs was decreased in c-Mpl knock-out HSCs (c-Mpl-KO). In addition, the MSCs co-cultured with c-Mpl-KO HSCs had reduced MSC marker expression (PDGFRa and Sca-1) compared to the MSCs co-cultured with c-Mpl-wild-type HSCs. These results suggest that a hematopoietic–mesenchymal signal exists, and that the state of the HSCs is important for the stability of MSC properties.     

MSC Pretreatment for Improved Transplantation Viability Results in Improved Ventricular Function in Infarcted Hearts

Many clinical studies utilizing MSCs (mesenchymal stem cells, mesenchymal stromal cells, or multipotential stromal cells) are underway in multiple clinical settings; however, the ideal approach to prepare these cells in vitro and to deliver them to injury sites in vivo with maximal effectiveness remains a challenge. Here, pretreating MSCs with agents that block the apoptotic pathways were compared with untreated MSCs. The treatment effects were evaluated in the myocardial infarct setting following direct injection, and physiological parameters were examined at 4 weeks post-infarct in a rat permanent ligation model. The prosurvival treated MSCs were detected in the hearts in greater abundance at 1 week and 4 weeks than the untreated MSCs. The untreated MSCs improved ejection fraction in infarcted hearts from 61% to 77% and the prosurvival treated MSCs further improved ejection fraction to 83% of normal. The untreated MSCs improved fractional shortening in the infarcted heart from 52% to 68%, and the prosurvival treated MSCs further improved fractional shortening to 77% of normal. Further improvements in survival of the MSC dose seems possible. Thus, pretreating MSCs for improved in vivo survival has implications for MSC-based cardiac therapies and in other indications where improved cell survival may improve effectiveness.     

Protective Effects of a Hyaluronan-Binding Peptide (P15-1) on Mesenchymal Stem Cells in an Inflammatory Environment

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1β)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.     

Mesenchymal Stem Cell-Based Therapy as an Alternative to the Treatment of Acute Respiratory Distress Syndrome: Current Evidence and Future Perspectives

Acute respiratory distress syndrome (ARDS) represents a current challenge for medicine due to its incidence, morbidity and mortality and, also, the absence of an optimal treatment. The COVID-19 outbreak only increased the urgent demand for an affordable, safe and effective treatment for this process. Early clinical trials suggest the therapeutic usefulness of mesenchymal stem cells (MSCs) in acute lung injury (ALI) and ARDS. MSC-based therapies show antimicrobial, anti-inflammatory, regenerative, angiogenic, antifibrotic, anti-oxidative stress and anti-apoptotic actions, which can thwart the physiopathological mechanisms engaged in ARDS. In addition, MSC secretome and their derived products, especially exosomes, may reproduce the therapeutic effects of MSC in lung injury. This last strategy of treatment could avoid several safety issues potentially associated with the transplantation of living and proliferative cell populations and may be formulated in different forms. However, the following diverse limitations must be addressed: (i) selection of the optimal MSC, bearing in mind both the heterogeneity among donors and across different histological origins, (ii) massive obtention of these biological products through genetic manipulations of the most appropriate MSC, (iii) bioreactors that allow their growth in 3D, (iv) ideal culture conditions and (v) adequate functional testing of these obtaining biological products before their clinical application.     

Dosing Limitation for Intra-Renal Arterial Infusion of Mesenchymal Stromal Cells

The immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) make MSC therapy a promising therapeutic strategy in kidney disease. A targeted MSC administration via the renal artery offers an efficient delivery method with limited spillover to other organs. Although local administration alleviates safety issues with MSCs in systemic circulation, it introduces new safety concerns in the kidneys. In a porcine model, we employed intra-renal arterial infusion of ten million allogenic adipose tissue-derived MSCs. In order to trigger any potential adverse events, a higher dose (hundred million MSCs) was also included. The kidney function was studied by magnetic resonance imaging after the MSC infusion and again at two weeks post-treatment. The kidneys were assessed by single kidney glomerular filtration rate (skGFR) measurements, histology and inflammation, and fibrosis-related gene expression. None of the measured parameters were affected immediately after the administration of ten million MSCs, but the administration of one hundred million MSCs induced severe adverse events. Renal perfusion was reduced immediately after MSC administration which coincided with the presence of microthrombi in the glomeruli and signs of an instant blood-mediated inflammatory reaction. At two weeks post-treatment, the kidneys that were treated with one hundred million MSCs showed reduced skGFR, signs of tissue inflammation, and glomerular and tubular damage. In conclusions, the intra-renal administration of ten million MSCs is well-tolerated by the porcine kidney. However, higher concentrations (one hundred million MSCs) caused severe kidney damage, implying that very high doses of intra-renally administered MSCs should be undertaken with caution.     

Addition of Adipose-Derived Stem Cells to Mesenchymal Stem Cell Sheets Improves Bone Formation at an Ectopic Site

To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration.     

Comparative Proteomic Analysis of the Mesenchymal Stem Cells Secretome from Adipose,Bone Marrow,Placenta and Wharton’s Jelly

Mesenchymal stem cells (MSCs) have the potential to be a viable therapy against various diseases due to their paracrine effects, such as secretion of immunomodulatory, trophic and protective factors. These cells are known to be distributed within various organs and tissues. Although they possess the same characteristics, MSCs from different sources are believed to have different secretion potentials and patterns, which may influence their therapeutic effects in disease environments. We characterized the protein secretome of adipose (AD), bone marrow (BM), placenta (PL), and Wharton’s jelly (WJ)-derived human MSCs by using conditioned media and analyzing the secretome by mass spectrometry and follow-up bioinformatics. Each MSC secretome profile had distinct characteristics depending on the source. However, the functional analyses of the secretome from different sources showed that they share similar characteristics, such as cell migration and negative regulation of programmed cell death, even though differences in the composition of the secretome exist. This study shows that the secretome of fetal-derived MSCs, such as PL and WJ, had a more diverse composition than that of AD and BM-derived MSCs, and it was assumed that their therapeutic potential was greater because of these properties.     

Intravascular Application of Labelled Cell Spheroids: An Approach for Ischemic Peripheral Artery Disease

Mesenchymal stem cells (MSC) are known for their vascular regeneration capacity by neoangiogenesis. Even though, several delivery approaches exist, particularly in the case of intravascular delivery, only limited number of cells reach the targeted tissue and are not able to remain on site. Applicated cells exhibit poor survival accompanied with a loss of functionality. Moreover, cell application techniques lead to cell death and impede the overall MSC function and survival. 3D cell spheroids mimic the physiological microenvironment, thus, overcoming these limitations. Therefore, in this study we aimed to evaluate and assess the feasibility of 3D MSCs spheroids for endovascular application, for treatment of ischemic peripheral vascular pathologies. Multicellular 3D MSC spheroids were generated at different cell seeding densities, labelled with ultra-small particles of iron oxide (USPIO) and investigated in vitro in terms of morphology, size distribution, mechanical stability as well as ex vivo with magnetic resonance imaging (MRI) to assess their trackability and distribution. Generated 3D spheroids were stable, viable, maintained stem cell phenotype and were easily trackable and visualized via MRI. MSC 3D spheroids are suitable candidates for endovascular delivery approaches in the context of ischemic peripheral vascular pathologies.     

The Secretome of Preconditioned Mesenchymal Stem Cells Drives Polarization and Reprogramming of M2a Macrophages toward an IL-10-Producing Phenotype

The preconditioning of mesenchymal stem cells (MSCs) has been recognized as an attractive tool to improve their regenerative and immunomodulatory capacities based on their paracrine effects. In this study, we examined the potential of an MSC-conditioned medium (MSC-CM) to alter the phenotype of murine macrophages and to drive reprogramming toward an anti-inflammatory, M2-like state in vitro. We further explored the impact of MSC cytokine preconditioning on the immunosuppressive properties of the MSC secretome. The MSC-CM suppressed the expression of proinflammatory genes in murine M1 macrophages, but only the CM from preconditioned MSCs (preMSC-CM) downregulated their expression during M1 polarization. Remarkably, only the preMSC-CM significantly increased the expression of M2a-, M2b- and M2c-specific genes and proteins during M2a polarization. Further, macrophages were found to secrete high levels of anti-inflammatory IL-10. Similarly, M2a macrophages cultured in the presence of the preMSC-CM displayed an enhanced expression of M2b/M2c-specific markers, suggesting that the secretome of preMSC promotes the repolarization of M2a-like macrophages to M2b/M2c subtypes. The preMSC-CM was found to be enriched in molecules involved in M2 polarization. Additionally, a unique downregulation of extracellular matrix components was observed. Altogether, the preMSC-CM may provide an attractive strategy to dampen inflammation by suppressing the expression of proinflammatory mediators and promoting the polarization and phenotype switch of M2a cells to IL-10-secreting M2b/M2c-like macrophages.     

Proteomic Comparison of Bone Marrow Derived Osteoblasts and Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, and therapeutic targeting of these cells is considered both for malignant and non-malignant diseases. We analyzed global proteomic profiles for osteoblasts derived from ten and MSCs from six healthy individuals, and we quantified 5465 proteins for the osteoblasts and 5420 proteins for the MSCs. There was a large overlap in the profiles for the two cell types; 156 proteins were quantified only in osteoblasts and 111 proteins only for the MSCs. The osteoblast-specific proteins included several extracellular matrix proteins and a network including 27 proteins that influence intracellular signaling (Wnt/Notch/Bone morphogenic protein pathways) and bone mineralization. The osteoblasts and MSCs showed only minor age- and sex-dependent proteomic differences. Finally, the osteoblast and MSC proteomic profiles were altered by ex vivo culture in serum-free media. We conclude that although the proteomic profiles of osteoblasts and MSCs show many similarities, we identified several osteoblast-specific extracellular matrix proteins and an osteoblast-specific intracellular signaling network. Therapeutic targeting of these proteins will possibly have minor effects on MSCs. Furthermore, the use of ex vivo cultured osteoblasts/MSCs in clinical medicine will require careful standardization of the ex vivo handling of the cells.     

Therapeutic Effect of Bone Marrow Mesenchymal Stem Cells on Laser-Induced Retinal Injury in Mice

Stem cell therapy has shown encouraging results for neurodegenerative diseases. The retina provides a convenient locus to investigate stem cell functions and distribution in the nervous system. In the current study, we investigated the therapeutic potential of bone marrow mesenchymal stem cells (MSCs) by systemic transplantation in a laser-induced retinal injury model. MSCs from C57BL/6 mice labeled with green fluorescent protein (GFP) were injected via the tail vein into mice after laser photocoagulation. We found that the average diameters of laser spots and retinal cell apoptosis were decreased in the MSC-treated group. Interestingly, GFP-MSCs did not migrate to the injured retina. Further examination revealed that the mRNA expression levels of glial fibrillary acidic protein and matrix metalloproteinase-2 were lower in the injured eyes after MSC transplantation. Our results suggest that intravenously injected MSCs have the ability to inhibit retinal cell apoptosis, reduce the inflammatory response and limit the spreading of damage in the laser-injured retina of mice. Systemic MSC therapy might play a role in neuroprotection, mainly by regulation of the intraocular microenvironment.     

Therapeutic Potential of Mesenchymal Stem Cells in a Pre-Clinical Model of Diabetic Kidney Disease and Obesity

Diabetic kidney disease (DKD) is a worldwide microvascular complication of type 2 diabetes mellitus (T2DM). From several pathological mechanisms involved in T2DM-DKD, we focused on mitochondria damage induced by hyperglycemia-driven reactive species oxygen (ROS) accumulation and verified whether mesenchymal stem cells (MSCs) anti-oxidative, anti-apoptotic, autophagy modulation, and pro-mitochondria homeostasis therapeutic potential curtailed T2DM-DKD progression. For that purpose, we grew immortalized glomerular mesangial cells (GMCs) in hyper glucose media containing hydrogen peroxide. MSCs prevented these cells from apoptosis-induced cell death, ROS accumulation, and mitochondria membrane potential impairment. Additionally, MSCs recovered GMCs’ biogenesis and mitophagy-related gene expression that were downregulated by stress media. In BTBR^ob/ob mice, a robust model of T2DM-DKD and obesity, MSC therapy (1 × 10⁶ cells, two doses 4-weeks apart, intra-peritoneal route) led to functional and structural kidney improvement in a time-dependent manner. Therefore, MSC-treated animals exhibited lower levels of urinary albumin-to-creatinine ratio, less mesangial expansion, higher number of podocytes, up-regulation of mitochondria-related survival genes, a decrease in autophagy hyper-activation, and a potential decrease in cleaved-caspase 3 expression. Collectively, these novel findings have important implications for the advancement of cell therapy and provide insights into cellular and molecular mechanisms of MSC-based therapy in T2DM-DKD setting.     

Silicon–Gold Nanoparticles Affect Wharton’s Jelly Phenotype and Secretome during Tri-Lineage Differentiation

Multiple studies have demonstrated that various nanoparticles (NPs) stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibit adipogenic ones. The mechanisms of these effects are not determined. The aim of this paper was to estimate Wharton’s Jelly MSCs phenotype and humoral factor production during tri-lineage differentiation per se and in the presence of silicon–gold NPs. Silicon (SiNPs), gold (AuNPs), and 10% Au-doped Si nanoparticles (SiAuNPs) were synthesized by laser ablation, characterized, and studied in MSC cultures before and during differentiation. Humoral factor production (n = 41) was analyzed by Luminex technology. NPs were nontoxic, did not induce ROS production, and stimulated G-CSF, GM-CSF, VEGF, CXCL1 (GRO) production in four day MSC cultures. During MSC differentiation, all NPs stimulated CD13 and CD90 expression in osteogenic cultures. MSC differentiation resulted in a decrease in multiple humoral factor production to day 14 of incubation. NPs did not significantly affect the production in chondrogenic cultures and stimulated it in both osteogenic and adipogenic ones. The major difference in the protein production between osteogenic and adipogenic MSC cultures in the presence of NPs was VEGF level, which was unaffected in osteogenic cells and 4–9 times increased in adipogenic ones. The effects of NPs decreased in a row AuNPs > SiAuNPs > SiNPs. Taken collectively, high expression of CD13 and CD90 by MSCs and critical level of VEGF production can, at least, partially explain the stimulatory effect of NPs on MSC osteogenic differentiation.     

Leukemia-Induced Cellular Senescence and Stemness Alterations in Mesenchymal Stem Cells Are Reversible upon Withdrawal of B-Cell Acute Lymphoblastic Leukemia Cells

Leukemic cell growth in the bone marrow (BM) induces a very stressful condition. Mesenchymal stem cells (MSC), a key component of this BM niche, are affected in several ways with unfavorable consequences on hematopoietic stem cells favoring leukemic cells. These alterations in MSC during B-cell acute lymphoblastic leukemia (B-ALL) have not been fully studied. In this work, we have compared the modifications that occur in an in vitro leukemic niche (LN) with those observed in MSC isolated from B-ALL patients. MSC in this LN niche showed features of a senescence process, i.e., altered morphology, increased senescence-associated β-Galactosidase (SA-βGAL) activity, and upregulation of p53 and p21 (without p16 expression), cell-cycle arrest, reduced clonogenicity, and some moderated changes in stemness properties. Importantly, almost all of these features were found in MSC isolated from B-ALL patients. These alterations rendered B-ALL cells susceptible to the chemotherapeutic agent dexamethasone. The senescent process seems to be transient since when leukemic cells are removed, normal MSC morphology is re-established, SA-βGAL expression is diminished, and MSC are capable of re-entering cell cycle. In addition, few cells showed low γH2AX phosphorylation that was reduced to basal levels upon cultivation. The reversibility of the senescent process in MSC must impinge important biological and clinical significance depending on cell interactions in the bone marrow at different stages of disease progression in B-ALL.