Murine Mesenchymal Stromal Cells Research Articles

Mesenchymal Stromal Cells Suppress T-Cell-Mediated Delayed-Type Hypersensitivity via ALCAM-CD6 Interaction.

Mounting evidence suggests mesenchymal stromal cells (MSCs) suppress CD4+ T-cell activation, but whether MSCs directly regulate activation and expansion of allogeneic T cells has not been fully deciphered. Here, we identified that both human and murine MSCs constitutively express ALCAM, a cognate ligand for CD6 receptors on T cells, and investigated its immunomodulatory function using in vivo and in vitro experiments. Our controlled coculture assays demonstrated that ALCAM-CD6 pathway is critical for MSCs to exert its suppressive function on early CD4+CD25- T-cell activation. Moreover, neutralizing ALCAM or CD6 results in the abrogation of MSC-mediated suppression of T-cell expansion. Using a murine model of delayed-type hypersensitivity response to alloantigen, we show that ALCAM-silenced MSCs lose the capacity to suppress the generation of alloreactive IFNγ-secreting T cells. Consequently, MSCs, following ALCAM knockdown, failed to prevent allosensitization and alloreactive T-cell-mediated tissue damage.

Bone marrow-derived extracellular vesicles carry the TGF-β signal transducer Smad2 to preserve hematopoietic stem cells in mice

SummaryExtracellular vesicles (EVs) released by cells in the bone marrow (BM) are important for regulating proliferation, differentiation, and other processes in hematopoietic stem cells (HSC). TGF-β signaling is now well known to be involved in HSC’s quiescence and maintenance, but the TGF-β pathway related to EVs is still largely unknown in the hematopoietic system. We found that the EV inhibitor Calpeptin, when injected intravenously into mice, particularly affected the in vivo production of EVs carrying phosphorylated Smad2 (p-Smad2) in mouse BM. This was accompanied with an alteration in the quiescence and maintenance of murine HSC in vivo. EVs produced by murine mesenchymal stromal MS-5 cells also showed presence of p-Smad2 as a cargo. We treated MS-5 cells with the TGF-β inhibitor SB431542 in order to produce EVs lacking p-Smad2, and discovered that its presence was required for ex vivo maintenance of HSC. In conclusion, we revealed a new mechanism involving EVs produced in the mouse BM that transport bioactive phosphorylated Smad2 as a cargo to enhance the TGF-β signaling-mediated quiescence and maintenance of HSC.

Impact of mesenchymal stromal cell-derived vesicular cargo on B-cell acute lymphoblastic leukemia progression.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Exvivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1β condition murine and human MSCs invitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.

Sickle Cell Disease Compromises the Hematopoietic Stem Cells Bone Marrow Niche with Age

Sickle cell disease (SCD) is characterized by chronic inflammation caused by red blood cell hemolysis, which can trigger oxidative stress and tissue injury that contribute to disease. Recent studies have shown that SCD pathophysiology reconstituted post-transplant in mouse models disrupts the architecture of bone marrow (BM) sinusoidal endothelium and causes a defect of mesenchymal stromal cells (MSCs) function. However, the impact of SCD on the ability MSCs and endothelial cells (ECs) to support of human and mouse hematopoiesis remains largely unknown. Using a mouse model of SCD, we now report that both sinusoidal (LepR+) and arteriolar MSCs (PαS) are more abundant in SCD BM, where they also display increased proliferation at 2-, 6- and 12-months of age. Additionally, 12-month-old SCD mice display a massive loss of sinusoidal ECs. Young SCD patient (n=9, age 2-18) BM also displayed increased MSC numbers relative to age-matched healthy controls (n=13). SCD MSCs isolated from 6- and 12-month old mice, maintained increased cell cycle when cultured ex vivo, showed decreased CFU-F potential, and impaired osteoblast differentiation. In ex vivo co-cultures of primary murine BM MSCs, ECs and HSCs, we found that 2-, 6-, and 12-month old SCD stroma (MSCs and ECs) failed to support non-SCD HSCs relative to controls. Phenotypic HSCs recovered from these co-cultures displayed a loss of CFU potential and transplantation activity. In tri-cultures, SCD MSCs, but not SCD ECs, drove increased HSC proliferation, apoptosis, and lymphoid-biased transplantation. In addition, MSCs isolated from BM of young SCD patients also displayed increased cell cycle, increased pro-inflammatory cytokine secretion, and decreased ability to support the expansion and CFU potential of co-cultured healthy human CD34+ cells, relative to MSCs isolated from young non-SCD donors. Bulk RNA-sequencing of 2-, 6-, and 12-month old murine SCD MSCs revealed gene signatures consistent with accelerated aging, increased proliferation, loss of extracellular matrix (ECM), impaired immune modulation, and reduced secretion of factors known to support HSCs. Genetic restoration of ECM rescued the ability of SCD MSCs to expand HSCs in ex vivo co-cultures, and support HSCs with robust CFU and transplantation potential. In sum, our data reveals a loss of HSPC supportive potential in BM MSCs in young individuals with SCD and that loss of ECM production by MSCs contributes to dysfunction in the HSC BM niche in both young and aged SCD mice.

The Role of Mitochondrial RNA from B-Precursor Acute Lymphoblastic Leukaemia Cells in Generating a Chemoprotective Bone Marrow Niche

Treatment resistant cells in precursor B-cell Acute Lymphoblastic Leukaemia (B-ALL) likely reside in a protective bone marrow niche with nestin-expressing mesenchymal stromal cells (MSC) (Duan et al Cancer Cell 2014). Previous work from our laboratory demonstrated that an 'activated MSC' niche, in which MSC have acquired features of cancer associated fibroblasts (CAF), develops in response to reactive oxygen species (ROS)-inducing chemotherapy. Mitochondria are actively transferred along tunneling nanotubes from activated MSC to B-ALL cells and can 'rescue' the cells from chemotherapy (Burt, et al Blood 2019). However, work from other groups suggests that chemoresistant ALL sub-clones exist prior to chemotherapy exposure, carrying a gene expression signature enriched for stemness, mitochondrial metabolism, stress responses and chromatin remodelling (Dobson et al Cancer Discovery 2020). We hypothesised that B-ALL cells may themselves directly activate bone marrow stromal cells and induce niche-formation prior to treatment, leading to ab-inito chemoresistance. To test this hypothesis, we first used our previously-described, in vitro niche model wherein either the HS27a MSC cell line or healthy donor MSCs were co-cultured with either B-ALL cell lines or primary patient B-ALL cells. Three of the five B-ALL cell lines and three of the four primary B-ALL samples that were tested induced activation of the MSCs, as characterised by cytomorphological changes identified by confocal microscopy, up-regulation of CAF-relevant gene expression and increased IL6, IL8 and CCL2 protein secretion. The ability of the B-ALL cell lines to activate MSC correlated directly with the intrinsic ROS level of the cell as determined by CellROX assay. Mitochondrial transfer from the cell-activated MSC to B-ALL cells, as measured by mitotracker dye transfer, occurred exactly as we had seen when we had used chemotherapy to activate the stroma. In vivo, the MSC-activating ALL cell lines also activated murine MSCs when established as xenografts in NOD-SCID gamma (NSG) mice. Immunohistochemistry of NSG murine femora revealed a very significant expansion in nestin+ cells after exposure to niche-induing ALL cells compared to non-niche-inducing controls. We aimed to determine the mechanism behind the B-ALL induced MSC activation. We determined that it was contact-independent, arising from exposure of MSC to ALL cells in transwell but also from exposure to B-ALL cell conditioned media alone. RNA sequencing of MSC after treatment with conditioned medium from activating B-ALL cells compared with non-activating controls unexpectedly showed that most highly upregulated pathways in the MSC-activating scenario were those involved in RNA sensing. Thus, we sought evidence for the role of mitochondrial dsRNA, as it had been recently shown to activate the anti-viral signalling pathway (Dhir et al, Nature 2018). Using the J2 anti-double stranded (ds) RNA monoclonal antibody in both confocal and flow cytometric assays, we observed very significantly higher levels of dsRNA in MSC which had been exposed to stromal-activating B-ALL cell conditioned medium than in non-activating B-ALL or MSC alone controls (see panel for image). We used the small molecule VBIT4, a voltage-dependent anion channel oligomerization inhibitor that decreases mitochondrial nucleic acid release to block this phenomenon in B-ALL cells. We quantified expression of a selection of stromal cell activation marker genes as well as genes in the RNA sensing pathway after treatment with VBIT4-exposed B-ALL cell CM or non-treated control. We observed a 2 to 4-fold reduction in expression of IL6 and CCL2 after VBIT4 exposure, compared to control. In the stromal cell RNA sensing pathway, there was a 15-fold reduction in expression of RIG-I and a 2 to 4-fold reduction in the interferon pathway genes MX2, IFIH1, MAVS and OAS1. Experiments to determine whether knock down of components of the antiviral sensing pathway also abrogates stromal activation and further murine experiments are ongoing. We propose a model in which ROS-induced mitochondrial dysfunction leads to release of dsRNA which in turn activates bone marrow stromal cells leading to protective niche formation and chemoprotection of acute lymphoblastic leukaemia cells despite lack of prior exposure to cytotoxic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Tissue Engineering Acoustophoretic (TEA) Set-up for the Enhanced Osteogenic Differentiation of Murine Mesenchymal Stromal Cells (mMSCs).

Quickly developing precision medicine and patient-oriented treatment strategies urgently require novel technological solutions. The randomly cell-populated scaffolds usually used for tissue engineering often fail to mimic the highly anisotropic characteristics of native tissue. In this work, an ultrasound standing-wave-based tissue engineering acoustophoretic (TEA) set-up was developed to organize murine mesenchymal stromal cells (mMSCs) in an in situ polymerizing 3-D fibrin hydrogel. The resultant constructs, consisting of 17 cell layers spaced at 300 µm, were obtained by continuous wave ultrasound applied at a 2.5 MHz frequency. The patterned mMSCs preserved the structured behavior within 10 days of culturing in osteogenic conditions. Cell viability was moderately increased 1 day after the patterning; it subdued and evened out, with the cells randomly encapsulated in hydrogels, within 21 days of culturing. Cells in the structured hydrogels exhibited enhanced expression of certain osteogenic markers, i.e., Runt-related transcription factor 2 (RUNX2), osterix (Osx) transcription factor, collagen-1 alpha1 (COL1A1), osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), as well as of certain cell-cycle-progression-associated genes, i.e., Cyclin D1, cysteine-rich angiogenic inducer 61 (CYR61), and anillin (ANLN), when cultured with osteogenic supplements and, for ANLN, also in the expansion media. Additionally, OPN expression was also augmented on day 5 in the patterned gels cultured without the osteoinductive media, suggesting the pro-osteogenic influence of the patterned cell organization. The TEA set-up proposes a novel method for non-invasively organizing cells in a 3-D environment, potentially enhancing the regenerative properties of the designed anisotropic constructs for bone healing.

P1246: DIFFERENTIAL ROLE OF MICROENVIRONMENT AND STROMAL-CELL INDUCED VENETOCLAX RESISTANCE IN MANTLE CELL LYMPHOMA

Background: Venetoclax is an inhibitor of the anti-apoptotic protein BCL-2 and a promising drug for MCL treatment. However, resistance towards venetoclax has already been reported in several MCL studies. There is growing evidence suggesting that interactions between lymphoma cells and their surrounding microenvironment might play a key role in not only promoting tumor cell survival but also facilitating resistance to certain chemotherapeutic agents, but little is known about the role of microenvironment in drug resistance in MCL. Aims: To understand the cellular and molecular resistance mechanisms mediated by the lymphoma microenvironment to prevent MCL patients from developing resistance to venetoclax treatment. To investigate the interactions of MCL cell lines with Mesenchymal Stromal Cells (MSCs), a key component of the microenvironment, in an in vitro co-culture system. Methods: MCL cell lines (MAVER-1, MINO) were cultured alone or in co-culture with HS-5, a human MSC cell line, or MS-5, a murine MSC cell line and exposed to different drugs, including venetoclax. Flow cytometry was used to assess cell viability. RNA sequencing of suspension and adherent MCL cells was used to identify differentially expressed genes. ELISA multiplex analysis was used to identify human or murine pro-inflammatory cytokines in co-culture supernatant. Results: Co-culture of MCL cell lines with both HS-5 and MS-5 induced resistance in MCL cells to doxorubicin and vincristine. However, only co-culture with MS-5 induced venetoclax resistance in MCL cells. Interestingly, conditioned media alone from MS-5 cultures was sufficient to induce venetoclax resistance, suggesting that soluble factors (such as cytokines and microvesicles) might mediate the venetoclax resistance mechanisms in MCL cells. Induction of apoptosis through MCL-1 inhibition with drug S6338 was also abrogated by co-culture with both HS-5 and MS-5, while BCL-xL inhibition using drug WEHI-549 was only ineffective in MINO cells in MS-5 co-culture. MCL cells in HS-5 co-cultures exhibited upregulation of genes involved in inflammatory response, cytokine signaling, and apoptosis, compared to MS-5 co-cultures. Accordingly, we identified unique sets of genes that were significantly upregulated in adherent MCL cells in HS-5 co-cultures, mainly involved in cell adhesion and cytokine signaling. Moreover, HS-5 co-cultures contained high levels of IL-6 and IL-8, corresponding with the increased gene expression levels of genes involved in the IL-6 signaling pathway in MCL cells. On the contrary, MS-5 co-cultures contained only high levels of murine KC/GRO, corresponding to human chemokine CXCL1. Image:Summary/Conclusion: We show that HS-5 provides MCL cells resistance to drugs, such as vincristine and doxorubicin, but not venetoclax, reflecting the different mechanisms of action of these therapies as well as their differential effects in the MCL microenvironment. MCL cells were resistant to venetoclax only in murine MSCs co-cultures, and further transcriptome and cytokine analysis supported this difference. Among others, our data questions how reliable murine MSCs are for obtaining translatable results, as MS-5 is the most used MSC cell line for co-cultures studies. These findings together provide a rationale to further investigate the role of MSCs in MCL drug resistance, as understanding the role of microenvironment in venetoclax sensitivity in MCL will potentially allow for more effective and targeted therapy.

Assessing the immunosuppressive activity of alginate-encapsulated mesenchymal stromal cells on splenocytes

Mesenchymal stromal cells (MSCs) show immunosuppressive effects both via cell-to-cell contact (direct) with immune cells and by producing paracrine factors and extracellular vesicles (indirect). A key challenge in delivering this therapeutic effect in vivo is retaining the MSCs at the site of injection. One way to address this is by encapsulating the MSCs within suitable biomaterial scaffolds. Here, we assess the immunosuppressive effect of alginate-encapsulated murine MSCs on proliferating murine splenocytes. Our results show that MSCs are able to significantly suppress splenocyte proliferation by ∼50% via the indirect mechanism and almost completely (∼98%) via the direct mechanism. We also show for the first time that MSCs as monolayers on tissue culture plastic or encapsulated within alginate, when physically isolated from the splenocytes via transwells, are able to sustain immunosuppressive activity with repeated exposure to fresh splenocytes, for as long as 9 days. These results indicate the need to identify design strategies to simultaneously deliver both modes of MSC immunosuppression. By designing cell-biomaterial constructs with tailored degradation profiles, we can achieve a more sustained (avoiding MSCs migration and apoptosis) and controlled release of both the paracrine signals and eventually the cells themselves enabling efficient MSC-based immunosuppressive therapies for wound healing.

Stem Cell Therapy Enhances Motor Activity of Triceps Surae Muscle in Mice with Hereditary Peripheral Neuropathy.

Impaired motor and sensory functions are the main features of Charcot–Marie–Tooth disease. Mesenchymal stem cell (MSCs) therapy is one of the possible treatments for this disease. It was assumed that MSCs therapy can improve the contractile properties of the triceps surae (TS) muscles in mice with hereditary peripheral neuropathy. Murine adipose-derived mesenchymal stromal cells (AD-MSCs) were obtained for transplantation into TS muscles of FVB-C-Tg(GFPU)5Nagy/J mice. Three months after AD-MSCs transplantation, animals were subjected to electrophysiological investigations. Parameters of TS muscle tension after intermittent high frequency electrical sciatic nerve stimulations were analyzed. It was found that force of TS muscle tension contraction in animals after AD-MSCs treatment was two-time higher than in untreated mice. Normalized values of force muscle contraction in different phases of electrical stimulation were 0.3 ± 0.01 vs. 0.18 ± 0.01 and 0.26 ± 0.03 vs. 0.13 ± 0.03 for treated and untreated animals, respectively. It is assumed that the two-fold increase in TS muscle strength was caused by stem cell therapy. Apparently, AD-MSCs therapy can promote nerve regeneration and partial restoration of muscle function, and thus can be a potential therapeutic agent for the treatment of peripheral neuropathies.

Lung Fibrosis Is Improved by Extracellular Vesicles from IFNγ-Primed Mesenchymal Stromal Cells in Murine Systemic Sclerosis.

Background: Systemic sclerosis (SSc) is a severe autoimmune disease for which mesenchymal stromal cells (MSCs)-based therapy was reported to reduce SSc-related symptoms in pre-clinical studies. Recently, extracellular vesicles released by MSCs (MSC-EVs) were shown to mediate most of their therapeutic effect. Here, we aimed at improving their efficacy by increasing the MSC-EV dose or by IFNγ-priming of MSCs. Methods: small size (ssEVs) and large size EVs (lsEVs) were recovered from murine MSCs that were pre-activated using 1 or 20 ng/mL of IFNγ. In the HOCl-induced model of SSc, mice were treated with EVs at day 21 and sacrificed at day 42. Lung and skin samples were collected for histological and molecular analyses. Results: increasing the dose of MSC-EVs did not add benefit to the dose previously reported to be efficient in SSc. By contrast, IFNγ pre-activation improved MSC-EVs-based treatment, essentially in the lungs. Low doses of IFNγ decreased the expression of fibrotic markers, while high doses improved remodeling and anti-inflammatory markers. IFNγ pre-activation upregulated iNos, IL1ra and Il6 in MSCs and ssEVs and the PGE2 protein in lsEVs. Conclusion: IFNγ-pre-activation improved the therapeutic effect of MSC-EVs preferentially in the lungs of SSc mice by modulating anti-inflammatory and anti-fibrotic markers.

Murine bone marrow mesenchymal stromal cells have reduced hematopoietic maintenance ability in sickle cell disease

AbstractSickle cell disease (SCD) is characterized by hemolytic anemia, which can trigger oxidative stress, inflammation, and tissue injury that contribute to disease complications. Bone marrow mesenchymal stromal cells (MSCs) tightly regulate hematopoietic stem cell (HSC) homeostasis in health and disease, but their functionality in SCD remains unclear. We identified for the first time that murine SCD MSCs have altered gene signatures, reduced stem cell properties, and increased oxidative stress, due in part to hemolysis. Murine SCD MSCs had lower HSC maintenance ability in vitro and in vivo, as manifested by increased HSC mobilization and decreased HSC engraftment after transplant. Activation of Toll-like receptor-4 through p65 in MSCs further contributed to MSC dysfunction. Transfusions led to an improved MSC and HSC oxidative state in SCD mice. Improving the regulation between MSCs and HSCs has vital implications for enhancing clinical HSC transplantation and gene therapy outcomes and for identification of new molecular targets for alleviating SCD complications.

The Intracerebroventricular Injection of Murine Mesenchymal Stromal Cells Engineered to Secrete Epidermal Growth Factor Does Not Prevent Loss of Neurogenesis in Irradiated Mice

Decreased neurogenesis after brain exposure to ionizing radiation is linked to neurocognitive impairments. Using transgenic mouse models, we previously showed that abrogation of radiation-induced senescence, or apoptosis, can partially rescue neurogenesis in the subventricular and hippocampus regions. Here, we evaluate whether the injection of recombinant epidermal growth factor (rEGF) or mesenchymal stromal cells (MSC) engineered to secrete EGF (MSC-EGF) can preserve neurogenesis. Using doublecortin (Dcx) expression and BrdU incorporation assays, we found that the injection of rEGF into the subventricular zone (SVZ) promotes neurogenesis, despite increasing apoptosis, in the brain of irradiated mice. The effect of rEGF was mostly localized, as Dcx expression was not induced in the hippocampus region and limited in the contralateral SVZ. Surprisingly, the injection of bone marrow-derived MSC alone, or secreting EGF, did not result in increased neurogenesis despite the fact that part of the MSC survived a few weeks after injection. Our results suggest that only a supraphysiological concentration of rEGF can promote neurogenesis, likely through a direct mitogenic effect.

Mesenchymal stromal cells-derived extracellular vesicles alleviate systemic sclerosis via miR-29a-3p

Systemic sclerosis (SSc) is a potentially lethal disease with no curative treatment. Mesenchymal stromal cells (MSCs) have proved efficacy in SSc but no data is available on MSC-derived extracellular vesicles (EVs) in this multi-organ fibrosis disease. Small size (ssEVs) and large size EVs (lsEVs) were isolated from murine MSCs or human adipose tissue-derived MSCs (ASCs). Control antagomiR (Ct) or antagomiR-29a-3p (A29a) were transfected in MSCs and ASCs before EV production. EVs were injected in the HOCl-induced SSc model at day 21 and euthanasized at day 42. We found that both ssEVs and lsEVs were effective to slow-down the course of the disease. All disease parameters improved in skin and lungs. Interestingly, down-regulating miR-29a-3p in MSCs totally abolished therapeutic efficacy. Besides, we demonstrated a similar efficacy of human ASC-EVs and importantly, EVs from A29a-transfected ASCs failed to improve skin fibrosis. We identified Dnmt3a, Pdgfrbb, Bcl2, Bcl-xl as target genes of miR-29a-3p whose regulation was associated with skin fibrosis improvement. Our study highlights the therapeutic role of miR-29a-3p in SSc and the importance of regulating methylation and apoptosis.

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.

Effect of hyaluronic acid on paracrine signaling of osteoblasts from mesenchymal stromal cells: potential impact on bone regeneration.

This study evaluated hyaluronic acids (HA) with different molecular weights as potential matrices for tissue-engineered bone grafting and their possible influence on the paracrine mechanisms of adipose-derived mesenchymal stromal cells. Murine adipose mesenchymal stromal cells (mASCs) on the fourth passage were seeded in 96-well plates, osteoinduced for 27 days and exposed for 3 days to low (HA-LW) and high/low molecular weight (HA-HLW) at previously defined concentrations. Cytokines IGF-1, VEGF, FGF-2, and BMP-2 were evaluated by quantification in the supernatant. Greater expression of growth factors was observed in groups with HA-HLW compared to HA-LW. Results indicated that differentiated cells secreted fewer cytokines, namely VEGF, FGF, and BMP-2 than undifferentiated mASCs (p < 0.05). IGF-1 showed its greatest expression in the mASC HA-LW group (p < 0.05). The application of HA-HLW as cell matrix in tissue engineering did not compromise mASC paracrine effect. Also, the association of HA-HLW matrix and mASCs resulted in greater expression of osteogenic growth factors. Longer periods of cell differentiation seemed to negatively affect their capacity for local paracrine stimulation. The use of HA-HLW as matrix for undifferentiated ASCs can be positive for bone regeneration, favoring its application as cell matrix in bone grafting procedures.

Hypoxia-cultured mouse mesenchymal stromal cells from bone marrow and compact bone display different phenotypic traits

It has been suggested that the bone marrow microenvironment harbors two distinct populations of mesenchymal stromal cells (MSC), one with a perivascular location and other present in the endosteum. A better understanding of the biology of these MSC subsets has been pursued in order to refine its clinical application. However, most comparative characterizations of mouse MSC have been performed in normoxia. This can result in misleading interpretations since mouse MSC subsets with low/defective p53 activity are known to be selected during culture in normoxia. Here, we report a comprehensive in vitro characterization of mouse MSC isolated from bone marrow (BM-MSC) and compact bone (CB-MSC) expanded and assayed under hypoxia for their morphology, clonogenic efficiency and differentiation capacity. We found that, under hypoxia, compact bone is richer in absolute numbers of MSC and isolation of MSC from compact bone is associated with a reduced risk of hematopoietic cell carryover. In addition, CB-MSC have higher in vitro osteogenic capacity than BM-MSC, while adipogenic differentiation potential is similar. These findings reinforce the hypothesis of the existence of MSC in bone marrow and compact bone representing functionally distinct cell populations and highlight the compact bone as an efficient source of murine MSC under physiological oxygen concentrations.

Evidence of mesenchymal stromal cell adaptation to local microenvironment following subcutaneous transplantation

Subcutaneous transplantation of mesenchymal stromal cells (MSC) emerged as an alternative to intravenous administration because it avoids the pulmonary embolism and prolongs post‐transplantation lifetime. The goal of this study was to investigate the mechanisms by which these cells could affect remote organs. To this aim, murine bone marrow–derived MSC were subcutaneously transplanted in different anatomical regions and the survival and behaviour have been followed. The results showed that upon subcutaneous transplantation in mice, MSC formed multicellular aggregates and did not migrate significantly from the site of injection. Our data suggest an important role of hypoxia‐inducible signalling pathways in stimulating local angiogenesis and the ensuing modulation of the kinetics of circulating cytokines with putative protective effects at distant sites. These data expand the current understanding of cell behaviour after subcutaneous transplantation and contribute to the development of a non‐invasive cell‐based therapy for distant organ protection.

Recent Approaches to Isolating and Culturing Mouse Bone Marrowderived Mesenchymal Stromal Stem Cells.

The isolation and culture of murine Bone Marrow-derived Mesenchymal stromal Stem Cells (mBMSCs) have attracted great interest in terms of the pre-clinical applications of stem cells in tissue engineering and regenerative medicine. In addition, culturing mBMSCs is important for studying the molecular mechanisms of bone remodeling using relevant transgenic mice. Several factors have created challenges in the isolation and high-yield expansion of homogenous mBMSCs; these factors include low frequencies of Bone Marrow-derived mesenchymal stromal Stem Cells (BMSCs) in bone marrow, variation among inbred mouse strains, contamination with Haematopoietic Progenitor Cells (HPCs), the replicative senescence phenotype and cellular heterogeneity. In this review, we provide an overview of nearly all protocols used for isolating and culturing mBMSCs with the aim of clarifying the most important guidelines for culturing highly purified mBMSC populations retaining in vitro and in vivo differentiation potential.

TGF-β1-Licensed Murine MSCs Show Superior Therapeutic Efficacy in Modulating Corneal Allograft Immune Rejection In Vivo

Mesenchymal stromal cells (MSCs) are a promising therapeutic option for multiple immune diseases/disorders; however, efficacy of MSC treatments can vary significantly. We present a novel licensing strategy to improve the immunosuppressive capacity of MSCs. Licensing murine MSCs with transforming growth factor-β1 (TGF-β MSCs) significantly improved their ability to modulate both the phenotype and secretome of inflammatory bone marrow-derived macrophages and significantly increased the numbers of regulatory T lymphocytes following co-culture assays. These TGF-β MSC-expanded regulatory T lymphocytes also expressed significantly higher levels of PD-L1 and CD73, indicating enhanced suppressive potential. Detailed analysis of T lymphocyte co-cultures revealed modulation of secreted factors, most notably elevated prostaglandin E2 (PGE2). Furthermore, TGF-β MSCs could significantly prolong rejection-free survival (69.2% acceptance rate compared to 21.4% for unlicensed MSC-treated recipients) in a murine corneal allograft model. Mechanistic studies revealed that (1) therapeutic efficacy of TGF-β MSCs is Smad2/3-dependent, (2) the enhanced immunosuppressive capacity of TGF-β MSCs is contact-dependent, and (3) enhanced secretion of PGE2 (via prostaglandin EP4 [E-type prostanoid 4] receptor) by TGF-β MSCs is the predominant mediator of Treg expansion and T cell activation and is associated with corneal allograft survival. Collectively, we provide compelling evidence for the use of TGF-β1 licensing as an unconventional strategy for enhancing MSC immunosuppressive capacity.

Nephroprotective Potential of Mesenchymal Stromal Cells and Their Extracellular Vesicles in a Murine Model of Chronic Cyclosporine Nephrotoxicity.

BackgroundCell therapies and derived products have a high potential in aiding tissue and organ repairing and have therefore been considered as potential therapies for treating renal diseases. However, few studies have evaluated the impact of these therapies according to the stage of chronic kidney disease. The aim of this study was to evaluate the renoprotective effect of murine bone marrow mesenchymal stromal cells (BM-MSCs), their extracellular vesicles (EVs) and EVs-depleted conditioned medium (dCM) in an aggressive mouse model of chronic cyclosporine (CsA) nephrotoxicity in a preventive and curative manner.MethodsAfter 4 weeks of CsA-treatment (75 mg/kg daily) mice developed severe nephrotoxicity associated with a poor survival rate of 25%, and characterized by tubular vacuolization, casts, and cysts in renal histology. BM-MSC, EVs and dCM groups were administered as prophylaxis or as treatment of CsA nephrotoxicity. The effect of the cell therapies was analyzed by assessing renal function, histological damage, apoptotic cell death, and gene expression of fibrotic mediators.ResultsCombined administration of CsA and BM-MSCs ameliorated the mice survival rates (6–15%), but significantly renal function, and histological parameters, translating into a reduction of apoptosis and fibrotic markers. On the other hand, EVs and dCM administration were only associated with a partial recovery of renal function or histological damage. Better results were obtained when used as treatment rather than as prophylactic regimen i.e., cell therapy was more effective once the damage was established.ConclusionIn this study, we showed that BM-MSCs induce an improvement in renal outcomes in an animal model of CsA nephrotoxicity, particularly if the inflammatory microenvironment is already established. EVs and dCM treatment induce a partial recovery, indicating that further experiments are required to adjust timing and dose for better long-term outcomes.