Two steps to functional mesenchymal stromal cells for clinical application

Long-term Engraftment of Multipotent Mesenchymal Stromal Cells That Differentiate to Form Myogenic Cells in Dogs With Duchenne Muscular Dystrophy

BackgroundThe treatment of tendon lesions with multipotent mesenchymal stromal cells (MSCs) is widely used in equine medicine. Cell sources of MSCs include bone marrow, as well as solid tissues such as adipose tissue. MSCs can be isolated from these solid tissues either by enzymatic digestion or by explant technique. However, the different preparation techniques may potentially influence the properties of the isolated MSCs. Therefore, the aim of this study was to investigate and compare the effects of these two different methods used to isolate MSCs from solid tissues.Equine adipose tissue, tendon and umbilical cord matrix served as solid tissue sources of MSCs with different stiffness and density. Subsequent to tissue harvest, MSCs were isolated either by enzymatic digestion with collagenase or by explant technique. Cell yield, growth, differentiation potential and tendon marker expression were analysed.ResultsAt first passage, the MSC yield was significantly higher in enzymatically digested tissue samples than in explanted tissue samples, despite a shorter period of time in primary culture. Further analysis of cell proliferation, migration and differentiation revealed no significant differences between MSCs isolated by enzymatic digestion and MSCs isolated by explant technique. Interestingly, analysis of gene expression of tendon markers revealed a significantly higher expression level of scleraxis in MSCs isolated by enzymatic digestion.ConclusionsBoth isolation techniques are feasible methods for successful isolation of MSCs from solid tissues, with no major effects on cellular proliferation, migration or differentiation characteristics. However, higher MSC yields were achieved in a shorter period of time by collagenase digestion, which is advantageous for the therapeutic use of MSCs. Moreover, based on the higher level of expression of scleraxis in MSCs isolated by enzymatic digestion, these cells might be a better choice when attempting tendon regeneration.

Genomic Stability and Safety of MSCs after Animal Serum-Free Humanized Clinical Scale Propagation.

To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony-forming unit-fibroblast (CFU-F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry-based methods for their evaluation. Pre-enrichment with D7-FIB-conjugated microbeads, cell sorting for CD45low D7-FIB+ LNGFR+ cells, and CFU-F assay were used to confirm the phenotype of BM MSCs in vivo. Further phenotypic characterization of MSCs was performed using three-color flow cytometry following pre-enrichment or by direct four-color flow cytometry. The sensitivity of direct flow cytometry/rare event analysis for the accurate enumeration of MSCs was validated using 85 samples from patients with neoplastic BM diseases. In normal BM, a significant correlation was found between the frequencies of CFU-Fs and CD45low D7-FIB+ LNGFR+ cells (n = 19, R = 0.719, P = 0.001). Following cell sorting, 15% of these cells were clonogenic. The same cells were enriched using LNGFR-based positive selection, CD45/Glycophorin A-based depletion, or plastic adherence. CD45low D7-FIB+ LNGFR+ cells expressed classic makers of cultured MSCs CD73/SH3 and CD105/SH2 and markers of stromal reticular cells CD106/VCAM and alkaline phosphatase. Novel markers were identified including leukemia inhibitory factor receptor and gp130. CD45low D7-FIB+ LNGFR+ cells were increased fourfold in the floating fat fraction of normal BM aspirates. Their frequency was decreased in chronic lymphocytic leukemia (threefold, n = 13, P = 0.049) and chronic myelogenous leukemia (ninefold, n = 11, P = 0.001) compared with that in age-matched controls (n = 26 and n = 31, respectively). This study demonstrates the usefulness of flow cytometry-based methods for the detection, enumeration and further phenotypic analysis of BM MSCs. These findings have broad applications for the future evaluation of BM MSCs in health and disease.

Changes in the Multipotent Stromal Mesenchymal Cells from the Bone Marrow of the Patients with Hematological Diseases in Debut and after the Treatment

Gap junctions (GJ) provide metabolic cooperation between cells through the direct exchange of cytoplasmic components. The authors analyzed the effect of short-term hypoxic stress on the efficiency of communication through the GJs in cultured multipotent mesenchymal stromal cells (MSCs) and characterized the sensitivity of MSCs to short-term hypoxic stress depending on the GJ function. Mitotically inactive MSCs were used in the experiments, in which the GJs were blocked with a specific inhibitor: carbenoxolone. The MSCs were continuously cultured at 20% O2. Further, MSCs with blocked and working GJs were subjected to hypoxic stress (0.1%, 24 h). The efficiency of GJ communication was attenuated under hypoxic stress. The combined action of GJ inhibition and hypoxic stress was accompanied by an increase in ROS level as compared to the MSCs after hypoxic stress only. MSCs with blocked GJs were less sensitive to short-term hypoxic stress in comparison with MSCs integrated into the common network through working GJs. This was manifested in attenuation of hypoxia-induced angiogenic activity of MSCs. The angiogenic effects of conditioned medium from the MSCs with blocked GJs were almost two times less, which seems to be related to differences in the angiogenic mediators’ profiles: VEGF level decreased and FGF-2 level increased, while the monocyte chemoattractant protein 3 (MCP-3) level was unchanged. Thus, a decrease in the efficiency of direct MSCs–MSCs communication had a negative effect on mostly requested MSC activity, the ability to induce angiogenesis. It is concluded that blocking of GJ communication in MSCs is a negative event that impairs the coordination of MSCs’ response to microenvironmental factors, in particular hypoxic stress, and reduces their functional plasticity.

Purpose Hyperthermia demonstrated clinical efficacy in multimodal cancer treatment. Multipotent mesenchymal stromal cells (MSCs) as part of the tumor-supporting stroma modulate tumor response and tissue regeneration after hyperthermia. We aimed to investigate the effects of hyperthermia on the survival, stem cell characteristics and heat shock expression of human MSCs. Materials and methods Human MSCs and normal human dermal fibroblasts (NHDFs) were exposed to temperatures between 37 °C and 44 °C for 60 min, and hyperthermic sensitivity was examined by clonogenicity, proliferation and viability assays. The influence of 42 °C hyperthermia on the MSCs’ adhesion potential, migratory capacity, surface marker expression and multi-lineage differentiation capability was investigated. Cell cycle distribution, apoptosis and senescence after 42 °C hyperthermia were determined by flow cytometry and β-galactosidase staining. Heat shock protein expression was determined by Western Blots. Results MSCs exhibited decreased clonogenic survival after 40 °C and 42 °C hyperthermia compared to NHDFs, while proliferative activity and viability were comparable after hyperthermia up to 44 °C. MSC adhesion was reduced after 42 °C hyperthermia, while the characteristic surface marker expression and the migratory ability remained unaffected in 42 °C hyperthermia-exposed MSCs. 42 °C hyperthermia diminished the adipogenic differential potential of all tested MSC samples. A pronounced G2/M arrest was found after 42 °C hyperthermia and was associated with increased apoptosis and senescence levels in MSCs. MSCs exhibited slightly lower heat shock protein levels compared to NHDFs. Conclusion Human MSCs exhibit a thermosensitive phenotype which reduced the multipotent cells’ regenerative abilities, resulting in impaired tissue regeneration after hyperthermia treatment or thermal injuries. On the other hand, tumor-associated MSCs may be efficiently targeted by hyperthermia treatment.

Bone marrow (BM) derived adult multipotent mesenchymal stromal cells (MMSCs) and fibroblast colony-forming units (CFU-Fs) of 20 patients with acute myeloid leukemia (AML) and 15 patients with acute lymphoblastic leukemia (ALL) before and during 1 year after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) were studied. The growth characteristics of MMSCs of all patients before allo-HSCT were not altered; however, relative expression level (REL) of some genes in MMSCs, but not in CFU-Fs, from AML and ALL patients significantly changed. After allo-HSCT, CFU-F concentration and MMSC production were significantly decreased for 1 year; REL of several genes in MMSCs and CFU-F-derived colonies were also significantly downregulated. Thus, chemotherapy that was used for induction of remission did not impair the function of stromal precursors, but gene expression levels were altered. Allo-HSCT conditioning regimens significantly damaged MMSCs and CFU-Fs, and the effect lasted for at least 1 year.

Advances in the field of Multipotent Mesenchymal Stromal cell (MSC) biology have demonstrated that MSCs can improve disease outcome when ‘activated' to exert immunomodulatory effects. However, the precise mechanisms modulating MSC-immune cells interactions remain largely elusive. In here, we activated MSC based on a recent polarization paradigm, in which MSCs can be polarized towards a pro- or anti-inflammatory phenotype depending on the Toll-like receptor stimulated, to dissect the mechanisms through which MSCs physically interact with and modulate leukocytes in this context. Our data show that MSCs activated through the Toll-like receptor (TLR) 4 pathway increased VCAM-1 and ICAM-1 dependent binding of leukocytes. On the other hand, TLR3 stimulation strongly increases leukocytes affinity to MSC comparatively, through the formation of cable-like hyaluronic acid structures. In addition, TLR4 activation elicited secretion of pro-inflammatory mediators by MSCs, whereas TLR3-activated MSCs displayed a milder pro-inflammatory phenotype, similar to inactivated MSCs. However, the differently activated MSCs maintained their ability to suppress leukocyte activation at similar levels in our in vitro model, and this immunomodulatory property was shown here to be partially mediated by prostaglandin. These results reinforce the concept that alternate activation profiles control MSC responses and may impact the therapeutic use of MSCs.

Multipotent mesenchymal stromal cell sheet therapy for bisphosphonate-related osteonecrosis of the jaw in a rat model

In vivo, bone marrow-derived multipotent mesenchymal stromal cells (MSC) have been identified at sites of tumors, suggesting that specific signals mobilize and activate MSC to migrate to areas surrounding tumors. The signals and migratory mechanisms that guide MSC are not well understood. Here, we investigated the migration of human MSC induced by conditioned medium of Huh-7 hepatoma cells (Huh-7 CM). Using a transwell migration system, we showed that human MSC migration was increased in the presence of Huh-7 CM. Using a human cytokine antibody array, we detected increased levels of MIP-1δ and MIP-3α in Huh-7 CM. Recombinant chemokines MIP-1δ and MIP-3α induced MSC migration. Anti-MIP-1δ and anti-MIP-3α antibodies added to Huh-7 CM decreased MSC migration, further suggesting that MIP-1δ and MIP-3α were implicated in the Huh-7 CM-induced MSC migration. By real-time polymerase chain reaction, we observed an absence of chemokine receptors CCR2 and CXCR2 and low expression of CCR1, CCR5, and CCR6 in MSC. Expression of these chemokine receptors was not regulated by Huh-7 CM. Furthermore, matrix metalloproteinase 1 (MMP-1) expression was strongly increased in MSC after incubation with Huh-7 CM, suggesting that MSC migration depends on MMP-1 activity. The signaling pathway MAPK/ERK was activated by Huh-7 CM but its inhibition by PD98059 did not impair Huh-7 CM-induced MSC migration. Further, long-term incubation of MSC with MIP-1δ increased α-smooth muscle actin expression, suggesting its implication in the Huh-7 CM-induced evolvement of MSC into myofibroblasts. In conclusion, we report that two inflammatory cytokines, MIP-1δ and MIP-3α, are able to increase MSC migration in vitro. These cytokines might be responsible for migration and evolvement of MSC into myofibroblasts around tumors.

Multipotent mesenchymal stromal cells (MSCs) have immunosuppressive capacity but the exact mechanism by which they suppress proliferation of T lymphocytes is not fully understood. Recently, the characteristics and function of regulatory T lymphocytes (Tregs) have become better defined. Tregs and MSCs have immunosuppressive features in common. Here, we looked for a common basis for immunosuppression in these distinct cell types. Forkhead box P3 (FoxP3) and CD39 expression in MSCs was measured by flow cytometry and real-time quantitative polymerase chain reaction. The importance of FoxP3 in MSC-mediated immunosuppression was investigated by siRNA technology and mixed lymphocyte culture (MLC). The effect of 5-azacytidine and other immunosuppressive drugs on FoxP3 expression and immunosuppression by MSCs was explored by flow cytometry, MLC, and real-time quantitative polymerase chain reaction. MSCs express FoxP3 at variable levels, but they do not express CD39. FoxP3 MSCs suppress MLC to a greater extent than cells with lower FoxP3 expression. However, FoxP3-decreased MSCs were found to retain their immunosuppressive properties. 5-azacytitine had no effect on FoxP3 expression or MLC suppression by MSCs. However, immunosuppressive drugs led to increased FoxP3 levels and MLC inhibition in FoxP3 MSCs. This is the first demonstration of FoxP3 expression by MSCs. Although MSCs share several features with Tregs, and FoxP3 MSCs tend to be more immunosuppressive, MSCs do not require functional FoxP3 for their immunosuppressive activity. The increased MSC-mediated suppression of immune responses by immunosuppressive drugs deserves further investigation.

Introduction. The properties of progenitor cells in the stromal microenvironment, i.e. multipotent mesenchymal stromal cells (MMSC) and fi broblast colony-forming units (CFU-F), undergo changes in patients with chronic myelogenous leukaemia (CML).Aim. To compare the progenitor cells of the stromal microenvironment (MMSCs and CFU-Fs) obtained from the bone marrow of CML patients at the onset of the disease, one year after the start of the treatment and during the long-term treatment with tyrosine kinase inhibitors (TKI).Materials and methods. The study involved an analysis of the characteristics of MMSCs, the concentration of CFU-Fs in the bone marrow of CML patients, as well as the relative expression level of genes (REL) associated with differentiation and involved in the regulation of haematopoiesis. The analysis was performed at the onset of the disease, one year after the start of the treatment, as well as 3–8 and 9–16 years after the TKI therapy. MMSCs and CFU-Fs of healthy donors were used for control purposes.Results. The concentration of CFU-Fs at the onset of the disease did not differ from that in donors; however, the relative expression level of genes associated with differentiation was increased in the CFU-F colonies. A year after the start of TKI treatment, the concentration of CFU-Fs decreased by four times. Subsequently, the concentration increased to reach normal values following 8 years of TKI treatment. The total production of MMSCs was not changed at the onset of the disease; however, it decreased after a year of TKI treatment, subsequently returning to normal. The expression of many genes was altered in the MMSCs of patients, i.e. the REL of LIF and JAG1 increased by 10 and 2 times, respectively; in the course of treatment, the REL of LIF in MMSCs decreased, always remaining higher than in those of the donors, whereas the expression of JAG1 returned to normal. At the onset of the disease, the REL of LIF in the MMSCs of patients, who achieved a deep molecular response (DMR) within 17 months of the treatment, was three times lower than in the MMSCs of those patients who did not reach DMR within 50 months, with JAG1 not differing from that of donors.Conclusion. Changes in stromal progenitor cells are associated with the influence of tumour cells, as well as with TKI therapy. A normal expression level of JAG1 and a decreased expression level of LIF in the MMSCs of CML patients at the onset of the disease may be predictive of DMR achievement.Conflict of interest: the authors declare no conflict of interest.Financial disclosure: the study had no sponsorship.

Multipotent mesenchymal stromal cells (MSCs) derived from adipose tissue have been increasingly studied due their lessinvasive extraction surgery, self-renew and differentiation potential. The literature has not been reported the comparison of different sources of MSCs derived from distinct adipose tissues and the use of electrical stimulation (ES) for tooth and bone tissue regeneration. The present study evaluated in vitro cell viability by MTT, cell proliferation by CCK-8 and the osteogenic differentiation by alizarin red-S staining of MSCs from Wistar rats adipose tissue, abdominal (ABD) and inguinal (ING), submitted to ES therapy. ES was applied during 60s, 150s and 300s at 10µA-intensity 3x/week. The ES therapy was not cytotoxic at any time of the experimentation. MSCs from ING presented higher percentage of cell viability at 60s of ES than MSCs from ABD stimulated for 150s and 300s. Moreover, MSCs from ING presented higher proliferation rate than ABD at all three ES time-application. The use of ES therapy during the osteogenic differentiation contributed positively to improve the mineralization process. MSCs from ING presented 30% greater mineralization at 150s, and 50% at 300s, while ABD showed 50% greater at 60s of ES. In conclusion, MSCs from ING and ABD are able to increase cell proliferation and improve osteogenic mineralization, but with different responses depending on the ES stimuli at the same experimental condition, which reinforce the needing for further investigation on the use of MSCs derived from fat tissue and ES to optimize their use for cell proliferation and mineralization in regenerative medicine.

Intrahippocampal transplantation of mesenchymal stromal cells promotes neuroplasticity