Irradiated Mesenchymal Stem Cells Research Articles

Non-small cell lung cancer cells and concomitant cancer therapy induce a resistance-promoting phenotype of tumor-associated mesenchymal stem cells.

The tumor microenvironment gained attraction over the last decades as stromal cells significantly impact on tumor development, progression and metastasis, and immune evasion as well as on cancer therapy resistance. We previously reported that lung-resident mesenchymal stem cells (MSCs) were mobilized and activated in non-small cell lung cancer (NSCLC) progression and could even mediate radiation resistance in co-cultured NSCLC cells. We investigated how MSCs were affected by NSCLC cells in combination with cancer (radiation) therapy in indirect co-cultures using tumor-conditioned medium and Transwells or direct three-dimensional NSCLC-MSC spheroid co-cultures in order to unravel the resistance-mediating action of tumor-associated MSCs. Although no obvious phenotypic and functional alterations in MSCs following NSCLC co-culture could be observed, MSC senescence was induced following co-applied radiotherapy (RT). Global gene expression profiling, in combination with gene set enrichment analysis upon treatment, was used to confirm the senescent phenotype of irradiated MSC and to reveal relevant senescence-associated secretory phenotype (SASP) factors that could meditate NSCLC RT resistance. We identified senescent tumor-associated MSC-derived serine proteinase inhibitor (serpin) E1/PAI1 as potential SASP factor mediating NSCLC progression and RT resistance. Specified intra-tumor-stroma interactions and cell type-specific pro-tumorigenic functions could not only improve lung cancer classification but could even be used for a more precise profiling of individual patients, finally paving an additional way for the discovery of potential drug targets for NSCLC patients.

Direct ionizing radiation and bystander effect in mouse mesenchymal stem cells

Purpose: This study aimed to evaluate the radiation-induced direct and bystander (BYS) responses of mesenchymal stem cells (MSCs) and to characterize these cells radiobiologically. Methods and materials: MSCs were irradiated (IR) and parameters related to DNA damage and cellular signaling were verified in a dose range from 0.5 to 15 Gy; also a transwell insert co-culture system was used to study medium-mediated BYS effects. Results: The main effects on directly IR cells were seen at doses higher than 6 Gy: induction of cell death, cell cycle arrest, upregulation of p21, and alteration of redox status. Irrespective of a specific dose, induction of micronuclei formation, H2AX phosphorylation, and decreased Akt expression also occurred. Thus, mTOR expression, cell senescence, nitric oxide generation, and calcium levels, in general were not significantly modulated by radiation. Data from the linear-quadratic model showed a high alpha/beta ratio, which is consistent with a more exponential survival curve. BYS effects from the unirradiated MSCs placed into companion wells with the directly IR cells, were not observed. Conclusions: The results can be interpreted as a positive outcome, meaning that the radiation damage is restricted to the directed IR MSCs not leading to off-target cell responses.

Mesenchymal stem cell-derived extracellular vesicles as probable triggers of radiation-induced heart disease

BackgroundRadiation-induced heart disease has been reported, but the underlying mechanisms remain unclear. Mesenchymal stem cells (MSCs), also residing in the heart, are highly susceptible to radiation. We examined the hypothesis that the altered secretion of extracellular vesicles (EVs) from MSCs is the trigger of radiation-induced heart disease.MethodsBy exposing human placental tissue-derived MSCs to 5 Gy γ-rays, we then isolated EVs from the culture medium 48 h later and evaluated the changes in quantity and quality of EVs from MSCs after radiation exposure. The biological effects of EVs from irradiated MSCs on HUVECs and H9c2 cells were also examined.ResultsAlthough the amount and size distribution of EVs did not differ between the nonirradiated and irradiated MSCs, miRNA sequences indicated many upregulated or downregulated miRNAs in irradiated MSCs EVs. In vitro experiments using HUVEC and H9c2 cells showed that irradiated MSC-EVs decreased cell proliferation (P < 0.01), but increased cell apoptosis and DNA damage. Moreover, irradiated MSC-EVs impaired the HUVEC tube formation and induced calcium overload in H9c2 cells.ConclusionsEVs released from irradiated MSCs show altered miRNA profiles and harmful effects on heart cells, which provides new insight into the mechanism of radiation-related heart disease risks.

The Role of Low-Dose Radiation in Association with TNF-α on Immunomodulatory Properties of Mesenchymal Stem Cells.

Ionizing radiation (IR) is an important medical tool. Despite the effects associated with high-dose radiation during or after treatment, as well as in accidental exposures, the direct or indirect effect of low-dose IR in cells remain poorly documented. IR can affect the tissue microenvironment, including mesenchymal stem cells (MSCs), which have high regenerative and immunomodulatory capacities. This study aimed to investigate the effect of low-dose IR in association with the inflammatory stimuli of TNF-α on the immunomodulatory capacity of MSCs. MSCs were irradiated with a low-dose IR, stimulated with TNF-α, and cultivated in a bystander system with murine spleen cells. The results showed that TNF-R1 is expressed in MSCs and is not affected, even in irradiated MSCs. However, irradiated MSCs produced reduced amounts of IL-6 and increased amounts of IL-10. The levels of PGE2 and NO• in MSCs were also increased when stimulated with TNF-α. Furthermore, conditioned media from irradiated MSCs reduced the proliferation of bystander lymphocytes and reduced the metabolic activity of macrophages. In addition, conditioned media from irradiated MSCs modulated the profile of cytokines in bystander spleen cells (lymphocytes and macrophages), reducing inflammatory and increasing anti-inflammatory cytokines, also increasing Treg cells. In conclusion, low-dose IR in association with an inflammatory stimulus affects the immunomodulatory properties of MSCs. In this way, the immunosuppressive capability of MSCs can be explored for several disease treatments where IR usually part of the context of the treatment. However, a complete understanding of the mechanisms underlying these interactions need further investigation. Graphical Abstract.

Irradiated mesenchymal stem cells support stemness maintenance of hepatocellular carcinoma stem cells through Wnt/\u03b2-catenin signaling pathway

BackgroundCancer stem cells are the main reason of relapse, metastasis and resistance to anti-cancer therapies of Hepatocellular carcinoma (HCC). Mesenchymal stem cells (MSCs) are an important part of the tumor microenvironment. MSCs have been demonstrated to be involved in drug resistance in tumor. How MSCs contribute to radiotherapy resistance of HCC is still indistinct.MethodsFlow cytometry analysis was performed to isolate CD133+ cells from HCC cell lines Huh7 and PLC. The stemness of Huh7-CD133 and PLC-CD133 those were co-cultured with IR-MSCs were investigated by Colony formation assay. Tumor formation in nude mice was used to explore the tumorigenicity of CD133+ cancer cells. The activating Wnt/β-catenin signaling pathway in CSCs were also detected by RT-PCR and Western blotting.ResultsWe report that irradiated MSCs (IR-MSCs) could increase the ratio of CD133+ cells in hepatocellular carcinoma cells. IR-MSCs could promote stemness maintenance of HCC stem cells. After co-cultured with IR-MSCs, liver cancer stem cells (CSCs) presented increased colony formation ability and tumor formation ability. We also found IR-MSCs promoted Wnt expression of CSCs. Reverse suppression experiment showed that when Wnt inhibitor was added into the culture medium, the effect of IR-MSCs on stemness maintenance was counteracted.ConclusionsThese data showed that IR-MSCs could support stemness maintenance of CSCs by activating Wnt/β-catenin signaling pathway.

Photopolymerizable biogel scaffold seeded with mesenchymal stem cells: safety and efficacy evaluation of novel treatment for intervertebral disc degeneration.

Tissue engineering approaches to treatment of intervertebral disc degeneration (IDD) represent a novel avenue of addressing the biologic basis of this disease. However, such approaches remain limited by their invasive nature and disruption to the annular fibrosis (AF). This study sought to explore a new minimally-invasive tissue-engineering approach utilizing an injection of a photopolymerizable biogel scaffold seeded with mesenchymal stem cells (MSCs) directly into the nucleus pulposus (NP). This study was performed using rabbit specimens for both in vivo and in vitro outcome measures. The treatment in this study was performed by injecting 25 μl of 10% (w/v) methacrylated gelatin biogel with 0.15% (w/v) lithium phenyl 2,4,6-trimethylbenzoylphosphinate (LAP) and rabbit MSCs (1 × 106 ) cells/ml into the NP. Samples were then photopolymerized in situ using non-ultraviolet light irradiation via a fiberoptic wire. For the in vitro arm of this study, gene expression analysis demonstrated increased anabolic activity in irradiated MSCs with and without biogel scaffolds. For the in vivo arm of this study, while GAG analysis did not demonstrate significant differences between groups, MRI analysis exhibited a trend toward improved NP matrix. Histological analysis was consistent with increased cellularity and less severe disc degeneration in the MSC + Gel group. However, osteophyte formation was noted in both Stab and MSC + Gel groups after the study period. Increased matrix gene expression of irradiated groups within in vitro studies indicates a photobiologic effect of 405 nm light. Despite promising anabolic actions, osteophyte formation and AF defects could not be avoided with implementation of this minimally-invasive tissue-engineering approach. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1451-1459, 2019.

Characteristics of tumors that have developed in mice injected with syngenic irradiated mesenchymal stem cells of bone marrow

Mesenchymal stem cells (MSCs) are found in virtually all organs and tissues. These cells can presumably be transformed into tumor stem cells by genotoxic factors and, subsequently, initiate tumor growth. The aim of the present work consisted in analysis of the possibility of malignant transformation of cultured MSCs from the bone marrow (BM) of mice after in vitro exposure to γ-radiation and in the characterization of biochemical and histological features of tumors that developed after the transplantation of BM MSCs to syngenic mice. Two of five mice developed tumors 3 to 4 months after the subcutaneous injection of BM MSCs irradiated at a dose of 1 Gy, five of five animals developed tumors after the administration of BM MSCs irradiated at a dose of 6 Gy, and only one of five mice injected with nonirradiated BM MSCs developed a tumor 6 months after cell transplantation. Telomerase activity in a tumor that developed from BM MSCs irradiated at a dose of 6 Gy was twice as high as that in the tumor that developed from BM MSCs irradiated at a dose of 1 Gy. The histological structure of the neoplasms corresponded to that of multicomponent mesenchymoma, a malignant tumor also termed “a mix of sarcomas.” The tumors consisted of tissue fragments of different histological types. Thus, BM MSCs exposed to 1 or 6 Gy of radiation can be transformed into tumor cells and give rise to multicomponent mesenchymomas, whereas malignant transformation of control BM MSCs occurs much less often.

Survivin Is a Master Regulator of Mesenchymal Stem Cell Functions and Potential Therapeutic Target for Mesenchymal Stem Cell Expansion

Mesenchymal stemcells (MSCs) are found throughout adult organisms and are involved in tissue maintenance and repair, as well as in the regulation of hematopoiesis and immunologic responses. Several clinical trials are currently under way to use allogeneic MSCs for enhancement of hematopoietic stem cell transplantation and treatment of graft-versus-host disease, spinal cord injury, cartilage and meniscus repair, and stroke. However, the therapeutic uses of MSCs can be limited by insufficient MSC number, their survival, and their ability to differentiate into multiple lineages, pointing to an important need to identify factors regulating MSC survival, proliferation, and differentiation. In this study, we explored the role of the anti-apototic protein Survivin in MSC functions. Survivin is an intracellular member of the inhibitor-of-apoptosis protein (IAP) family and is implicated in regulation of apoptosis, cell division, and cell cycle control of many cell types including hematopoietic stem cells, leukemic cells, and endothelial cells. However, whether Survivin is involved in MSC activity or function is unknown. Using flow cytometry and function assays, we found that adult mouse and human MSCs, identified as CD45-Ter119-CD51+PDGFR+Nestin+ cells, express high levels of Survivin, which regulates MSCs survival and expansion under normal physiological and stressful conditions. Flow cytometry analysis revealed that approximately 40.5±3.6% of mouse bone marrow MSCs, 33.6±4.2% of adult human bone marrow MSCs, and 43.6±5.3% human cord blood MSCs express Survivin. Treatment of mice in vivo with YM155 (10 mg/kg), a small molecule Survivin inhibitor, for 6 days decreased bone marrow CD45-Ter119-CD51+PDGFR+Nestin+ MSC by 2.1 fold and functional fibroblast colony formation (CFU-F) by 2.8 fold. Survivin gene deletion using Survivin-specific SiRNA decreased mouse and human bone marrow derived MSCs number and CFU-F ability by 3.2 fold and 2.8 fold respectively. Retroviral overexpression of Survivin in mouse MSCs enhanced CFU-F formation by 4 fold. In an in vitro wound healing assay YM155-treated MSCs recovered more slowly compared to control cultures. To determine underlying mechanisms involved in Survivin dependent regulation of MSC function, we measured the survival and proliferation of YM155 treated MSCs. Treatment of mouse or human bone marrow-derived MSCs with YM155 (50 nmol/l) for 48 hours enhanced the caspase 3 and 7 expression by 42.8% and 63.9% respectively, while BrdU incorporation was similar in control and YM155-treated MSCs, suggesting that Survivin is primarily involved in MSC survival. To explore whether Survivin is also involved in growth factor-mediated MSC survival and expansion, mouse bone marrow derived MSCs were cultured in the presence of platelet derived growth factor (PDGF) or basic fibroblast growth factor (b-FGF) with or without the Survivin inhibitor YM155. PDGF enhanced the Survivin expression by 52.8±2.2% and b-FGF enhanced Survivin expression by 43.8±4.5% and increased the CFU-F counts by 5.6 fold and 4.2 fold, respectively. In contrast, YM155 treated MSCs did not show any enhancement in Survivin expression, and CFU-F were substantially lower than the corresponding controls (PDGF or b-FGF treated cultures). To determine whether Survivin affects the Survival of stressed MSCs, we exposed mouse bone marrow MSCs to 4 Gy irradiation dose and followed by culture for 3 days with or without the Survivin inhibitor YM155. Irradiation alone reduced the survival of MSCs by 53.5%, however the viability of irradiated MSCs treated with YM155 was reduced by 82.8%. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We found that the exposure of MSC cultures to YM155 during the lineage differentiation process inhibits MSC osteogenic and adipogenic differentiation while sparing chondrogenic cell differentiation. In conclusion, our study demonstrates that Survivin controls basal and growth factor dependent survival and expansion of mouse and human MSCs, and protects them from irradiation induced cell death. Furthermore, our data suggest the regulating Survivin expression in MSCs would be beneficial to enhance MSC recovery for clinical utility. Disclosures No relevant conflicts of interest to declare.

The effect of ATM kinase inhibition on the initial response of human dental pulp and periodontal ligament mesenchymal stem cells to ionizing radiation

Purpose: This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pulp and periodontal ligament after γ-irradiation and the effect of ataxia-telangiectasia mutated (ATM) inhibition.Methods: MSC were irradiated with 2 and 20 Gy by 60Co. For ATM inhibition, specific inhibitor KU55933 was used. DNA damage was measured by Comet assay and γH2AX detection. Cell cycle distribution and proteins responding to DNA damage were analyzed 2–72 h after the irradiation.Results: The irradiation of MSC causes an increase in γH2AX; the phosphorylation was ATM-dependent. Irradiation activates ATM kinase, and the level of p53 protein is increased due to its phosphorylation on serine15. While this phosphorylation of p53 is ATM-dependent in MSC, the increase in p53 was not prevented by ATM inhibition. A similar trend was observed for Chk1 and Chk2. The increase in p21 is greater without ATM inhibition. ATM inhibition also does not fully abrogate the accumulation of irradiated MSC in the G2-phase of the cell-cycle.Conclusions: In irradiated MSC, double-strand breaks are tagged quickly by γH2AX in an ATM-dependent manner. Although phosphorylations of p53(ser15), Chk1(ser345) and Chk2(thr68) are ATM-dependent, the overall amount of these proteins increases when ATM is inhibited. In both types of MSC, ATM-independent mechanisms for cell-cycle arrest in the G2-phase are triggered.

Insulin-like growth factor binding protein-2 and neurotrophin 3 synergize together to promote the expansion of hematopoietic cells ex vivo

Co-culture of Umbilical Cord Blood (UCB) CD34+ cells with irradiated Mesenchymal Stem Cells (MSCs) without contact increase the expansion of Hematopoietic Progenitor Cells (HPC). Neurotrophin-3 (NT-3) and insulin-like growth factor binding protein-2 (IGFBP-2) are two factors whose expressions were significantly elevated in conditioned media derived from irradiated MSCs. To determine whether these factors are partly responsible for the growth promoting potential of MSCs, we investigated their impact on the growth and differentiation of UCB-CD34+ cells. Addition of either factor alone had little impact on cell growth, however both factors synergized together to increase the expansion of total nucleated cells, erythroids, megakaryocytes (Mk) and CD34+ cells. However, in contrast to MSCs they failed to significantly improve the expansion of hematopoietic progenitors. Consistent with the impact of these factors on hematopoietic cells, both synergized to activate ERK1/2 and AKT in primary human UCB cells. In conclusion, the study demonstrates for the first time that a neurotrophin factor can synergize with IGFBP-2 to promote hematopoietic cell expansion.

Irradiated mesenchymal stem cells improve the ex vivo expansion of hematopoietic progenitors by partly mimicking the bone marrow endosteal environment

Mesenchymal Stem Cells (MSCs) regulate the growth and differentiation of Hematopoietic Progenitor cells (HPCs) through the release of soluble factors or through their differentiation into osteoblasts. We recently demonstrated that expansion of megakaryocyte (MK) progenitors ex vivo had reached a plateau when CD34 + cells were grown with two optimized cytokine cocktails developed for the growth of MK. Hence, we sought to determine whether co-culture of CD34 + cells with Bone Marrow (BM) MSCs could further increase the expansion of myeloid and MK progenitors. First, we tested the impact of cell–cell contact and pre-irradiation treatment of the MSCs to identify the condition that best supports HPC expansion. This screen revealed that HPC expansions were generally greater in the non-contact conditions, and that pre-irradiation of the MSCs appeared to be of added benefits. Improved expansion of both myeloid and MK progenitors in co-culture with irradiated MSCs without contact was subsequently confirmed. Next, cytokine array profiling was carried out to investigate why irradiation promoted progenitor expansion. This revealed that the levels of as many as 33 factors were potentially altered. ELISA confirmed the significant up regulation of NT-3 and IGFBP-2. Since, these factors are known to be released by and important for osteogenic and endothelial cells, we investigated and confirmed that irradiation of MSCs induced their rapid differentiation into osteogenic-like cells, but not into endothelial-like cells. Supporting this finding, expansions of myeloid and MK progenitors were increased when CD34 + cells were co-culture with MSCs-derived osteoblasts. Altogether, these results indicate that the improved expansion of HPCs obtained with irradiated MSCs is due in part to their differentiation into osteoblast-like cells, thereby recreating an endosteal-like environment that provides improved support for HPCs expansion.

Mesenchymal stem cells protect NOD mice from diabetes by inducing regulatory T cells

Displaying immunomodulatory capacities, mesenchymal stem cells (MSCs) are considered as beneficial agents for autoimmune diseases. The aim of this study was to examine the ability of MSCs to prevent autoimmune diabetes in the NOD mouse model. Prevention of spontaneous insulitis or of diabetes was evaluated after a single i.v. injection of MSCs in 4-week-old female NOD mice, or following the co-injection of MSCs and diabetogenic T cells in irradiated male NOD recipients, respectively. The frequency of CD4(+)FOXP3(+) cells and Foxp3 mRNA levels in the spleen of male NOD recipients were also quantified. In vivo cell homing was assessed by monitoring 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T cells or MSCs. In vitro, cell proliferation and cytokine production were assessed by adding graded doses of irradiated MSCs to insulin B9-23 peptide-specific T cell lines in the presence of irradiated splenocytes pulsed with the peptide. MSCs reduced the capacity of diabetogenic T cells to infiltrate pancreatic islets and to transfer diabetes. This protective effect was not associated with the modification of diabetogenic T cell homing, but correlated with a preferential migration of MSCs to pancreatic lymph nodes. While injection of diabetogenic T cells resulted in a decrease in levels of FOXP3(+) regulatory T cells, this decrease was inhibited by MSC co-transfer. Moreover, MSCs were able to suppress both allogeneic and insulin-specific proliferative responses in vitro. This suppressive effect was associated with the induction of IL10-secreting FOXP3(+) T cells. MSCs prevent autoimmune beta cell destruction and subsequent diabetes by inducing regulatory T cells. MSCs may thus offer a novel cell-based approach for the prevention of autoimmune diabetes and for islet cell transplantation.

Implantation of Low-Level Laser Irradiated Mesenchymal Stem Cells into the Infarcted Rat Heart Is Associated with Reduction In Infarct Size and Enhanced Angiogenesis

The aim of the present study was to evaluate the possible beneficial effects of implantation of laser-irradiated mesenchymal stem cells (MSCs) into the infarcted rat heart. It was demonstrated that low-level laser therapy (LLLT) upregulates cytoprotective factors in ischemic tissues. MSCs were isolated from rat bone marrow and grown in culture. The cells were laser irradiated with a Ga-Al-As laser (810 nm wavelength), labeled with 5-bromo-2'deoxyuridine (BrdU), and then implanted into infarcted rat hearts. Non-irradiated cells were similarly labeled and acted as controls. Hearts were excised 3 wk later and cells were stained for BrdU and c-kit immunoreactivity. Infarcted hearts that were implanted with laser-treated cells showed a significant reduction of 53% in infarct size compared to hearts that were implanted with non-laser-treated cells. The hearts implanted with laser-treated cells prior to implantation demonstrated a 5- and 6.3-fold significant increase in cell density that positively immunoreacted to BrdU and c-kit, respectively, as compared to hearts implanted with non-laser-treated cells. A significantly 1.4- and 2-fold higher level of angiogenesis and vascular endothelial growth factor, respectively, were observed in infarcted hearts that were implanted with laser-treated cells compared to non-laser-treated implanted cells. The findings of the present study provide the first evidence that LLLT can significantly increase survival and/or proliferation of MSCs post-implantation into the ischemic/infarcted heart, followed by a marked reduction of scarring and enhanced angiogenesis. The mechanisms associated with this phenomenon remain to be elucidated in further studies.

Soluble mediators from mesenchymal stem cells suppress T cell proliferation by inducing IL-10

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either na?ve or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naive and pre-activated T cells in which IL-10 and IDO play important roles.

Human Mesenchymal Stem Cells Exert Immunosuppressive Capacities by Induction of Regulatory T Cells

It is widely accepted that Mesenchymal Stem Cells (MSCs) exhibit immunosuppressive capacities in vitro and in vivo. In an effort to understand the mechanism of suppression, co-cultures of MSCs with several types of immune cells have been studied. MSCs inhibit B- and T cell proliferation and inhibit the generation, maturation and function of dendritic cells. In the present study, we evaluated the effect of MSCs on the expansion and function of regulatory T cells (T regs). Human bone-marrow derived MSCs were cultured and expanded in low-glucose DMEM containing 10% FCS and frozen until used in experiments. Freshly isolated, MACS selected human CD4+CD25+ T regs were cultured in IMDM, supplemented with 10% pooled human serum and 300 units/ml IL-2, in the presence or absence of irradiated MSCs (60 Gy) (ratio MSCs: T regs = 1:5). Results are derived from 3 to 15 independent experiments. In the presence of IL-2 and MSCs, the percentage of FOXP3+ CD4+CD25+ T cells increased from 26.8% ±2.2 (no MSCs) to 42.3% ±2.4 (with MSCs) over a period of 5 days, representing a 1.6 (±0.1)-fold induction. Moreover, a distinct CD4+CD25+ population with high FOXP3 expression appeared after 5 days of culture in the presence of MSCs (23.3% ±2.5 in the presence of MSCs versus 7.2% ±1.0 in the absence of MSCs). This CD4+CD25+FOXP3hi population was not observed after co-culturing MSCs and CD4+CD25− T cells (1.7% ±0.6). To show that the MSC-induced T regs were functionally suppressive, freshly isolated CFSE-labeled CD4+ T cells were stimulated with PHA (0.8 μg/ml). After 3 days, about 40% of the T cells had undergone one or more cell divisions as measured by CFSE dilution. Addition of MSC-induced T regs (in a 1:1 ratio) resulted in a 50% reduction of the proliferation of CFSE-labeled T cells (down to 15–20% of cells undergoing one or more cell divisions). Control CD4+CD25+ T cells cultured in the absence of MSCs did not suppress T cell proliferation. These results indicate that MSC-induced CD4+CD25+ FOXP3hi cells exert regulatory function. To study whether the induction of T regs by MSCs was dependent on cell-cell contact, co-culture experiments were performed in transwells where MSCs were physically separated from T cells. CD4+CD25+ T cells co-cultured with MSCs in transwells showed a 1.4 (±0.1)-fold increase in the percentage of FOXP3hi cells, in comparison with T cells cultured in the absence of MSCs. Similarly, direct co-cultures of MSCs and T cells resulted in a 1.8 (±0.2)-fold increase in the percentage of FOXP3hi T regs. In addition, medium derived from co-cultures of MSCs and CD4+CD25+ T cells and added to freshly isolated CD4+CD25+ T cells resulted in a 2.6 (±0.6) fold increase in the percentage of CD4+CD25+FOXP3hi T regs, implicating that FOXP3hi induction by MSCs was mediated by soluble factors. Since T Cell Receptor-stimulated naïve T cells (as well as natural T regs, generated in the thymus) require both IL-2 and TGF-β to become induced T regs, we analyzed the involvement of TGF-β. Addition of the pharmacological inhibitor of the TGF-β receptor (SB431542) only marginally reduced FOXP3 induction in the presence of MSCs, suggesting that MSC-mediated expression of high FOXP3 levels requires alternate or additional cytokines. In conclusion, we show thatMSCs promote the induction of CD4+CD25+ T cells that express high levels of FOXP3 andthese MSC-induced T regs suppress proliferation of PHA-stimulated CD4+ T cells.These effects are mediated by soluble factors produced during the co-culture of MSCs and T cells. The cytokines involved are presently unknown, but likely do not involve TGF-β.

Ionizing Radiation of Mesenchymal Stem Cells Results in Diminution of the Precursor Pool and Limits Potential for Multilineage Differentiation

Although ionizing radiation is an important treatment modality for a number of malignancies, it can be associated with significant morbidity. The exact mechanisms by which ionizing radiation results in cellular injury remain unknown. Mesenchymal stem cells give rise to a number of tissues including bone, fat, and cartilage and provide an excellent cellular model with which to evaluate the effects of ionizing radiation on cellular survival and function. Rat mesenchymal stem cells were irradiated with 0, 7, and 12 Gy of ionizing radiation and assessed for changes in growth, apoptosis, cell-cycle profile, senescence, differentiation, and gene expression. Ionizing radiation resulted in a significant decrease in cellular proliferation because of increased apoptosis, G2 cell-cycle arrest, and premature senescence. In addition, ionizing radiation caused low-level spontaneous osteoblastic differentiation. Conversely, cellular differentiation in response to lineage-specific culture conditions for bone, fat, and cartilage was markedly decreased in irradiated cells, thereby demonstrating a deficit in the ability of irradiated mesenchymal stem cells to respond to environmental stimuli. Although the majority of mesenchymal stem cells survive injury from ionizing radiation, this injury results in a significant decrease in cellular proliferation. Furthermore, the differentiation potential of irradiated mesenchymal stem cells in response to environmental stimuli is markedly diminished. Thus, the negative effects of ionizing radiation may result from a decreased pool of progenitor cells with limited differentiation potential. Proposed radioprotection strategies aiming to reduce tissue injury should therefore evaluate not only cellular survival but also cellular function.

Low level laser irradiation stimulates osteogenic phenotype of mesenchymal stem cells seeded on a three-dimensional biomatrix

Mesenchymal stem cells (MSCs) seeded on three-dimensional (3D) coralline (Porites lutea) biomatrices were irradiated with low-level laser irradiation (LLLI). The consequent phenotype modulation and development of MSCs towards ossified tissue was studied in this combined 3D biomatrix/LLLI system and in a control group, which was similarly grown, but was not treated by LLLI. The irradiated and non irradiated MSC were tested at 1-7, 10, 14, 21, 28 days of culturing via analysis of cellular distribution on matrices (trypan blue), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregates formation (oil red O), alkaline phosphatase (ALP) activity, scanning electron microscopy (SEM) and electron dispersive spectrometry (EDS). The results obtained from the irradiated samples showed enhanced tissue formation, appearance of phosphorous peaks and calcium and phosphate incorporation to newly formed tissue. Moreover, in irradiated samples ALP activity was significantly enhanced in early stages and notably reduced in late stages of culturing. These findings of cell and tissue parameters up to 28 days of culture revealed higher ossification levels in irradiated samples compared with the control group. We suggest that both the surface properties of the 3D crystalline biomatrices and the LLLI have biostimulatory effects on the conversion of MSCs into bone-forming cells and on the induction of ex-vivo ossification.

Human mesenchymal stem cells affect IgG production induced by lipopolysaccharide, cytomegalovirus and varicella zoster virus in human spleen cells

Human mesenchymal stem cells (MSCs) suppress T-cell proliferation and formation of cytotoxic T lymphocytes in vitro. In vivo, MSCs prolong skin allograft survival and mitigate severe graft-versus-host disease. The immunomodulatory properties of MSCs have focused on T cells. We have examined the effects of MSCs on human splenic B-cell IgG secretion. IgG response was analyzed using the ELI-spot assays, after stimulating of human spleen cells with lipopolysaccaride (LPS), cytomegalovirus (CMV), or varicella zoster virus (VZV) antigens with or without 10% irradiated MSCs.

Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells.

Mesenchymal stem cells (MSCs) can reduce the incidence of graft-versus-host disease because of their ability to inhibit T-lymphocyte proliferation. There are no publications on the effect that MSCs have on cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, effector cells vital for the graft-versus-leukemia effect. Cytotoxic T cells were primed in mixed lymphocyte culture (MLC) against irradiated stimulator lymphocytes, and irradiated third-party MSCs were added at different time points. The CTLs were collected, and their cytotoxic potential was analyzed in a chromium-release assay against the same stimulator cells as in the MLC. Purified NK cells were mixed with irradiated MSCs, and the lysis was measured in chromium-release assay against K562 target cells. We found that MSCs inhibited CTL-mediated lysis by 70% if added at the beginning of the 6-day MLC. The lysis was not affected on day 3 or in the cytotoxic phase. Furthermore, MSCs inhibited the formation of cytotoxic lymphocytes when the cells were separated in a transwell system, which indicates that the effect is mediated by a soluble factor. NK cell-mediated lysis of K562 cells was not inhibited by MSCs. MSCs did not induce proliferation of allogeneic lymphocytes, and they were not lysed by allogeneic CTLs or NK cells. Our findings indicate that MSCs escape recognition by CTLs and alloreactive NK cells, and inhibit the formation of cytotoxic T cells by secreting a soluble factor, but that they do not interfere with CTLs and NK cell lysis.

HLA expression and immunologic propertiesof differentiated and undifferentiated mesenchymal stem cells

Objective Mesenchymal stem cells (MSC) do not elicit alloreactive lymphocyte responses due to immune modulations. We investigated the immunologic properties of MSC after differentiation along three lineages: bone, cartilage, and adipose. Methods and results Flow cytometry showed that undifferentiated MSC express HLA class I but not class II, although HLA class II was present intracellularly as detected by Western blot. Addition of interferon γ (IFN-γ) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic irradiated MSC as stimulators. Results were similar using MSC pretreated with IFN-γ. After growth of cells in medium to induce differentiation to bone, cartilage, or adipose for 6 or 12 days, the expression of HLA class I increased but no class II was detected on the cell surface. The ability to upregulate HLA class II on the cell surface after exposure to IFN-γ for 48 hours was clearly diminished after the cells had been cultured in differentiation medium for 6 or 12 days, with only 10% of cells expressing HLA class II. Using MSC grown in osteogenic, chondrogenic, or adipogenic medium as stimulator cells, no lymphocyte alloreactivity was seen, even if differentiated MSC had been pretreated with IFN-γ. MSC inhibit mixed lymphocyte cultures, particularly after osteogenic differentiation. This suppression was further enhanced by IFN-γ. Conclusion Undifferentiated and differentiated MSC do not elicit alloreactive lymphocyte proliferative responses and modulate immune responses. The findings support that MSC can be transplantable between HLA-incompatible individuals.