Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model

Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions.

Characterization of a stem cell population in lung cancer A549 cells

We isolated a stem cell subpopulation from human lung cancer A549 cells using FACS/Hoechst 33342. This side population (SP), which comprised 24% of the total cell population, totally disappeared after treatment with the selective ABCG 2 inhibitor fumitremorgin C. In a repopulation study, isolated SP and non-SP cells were each able to generate a heterogeneous population of SP and non-SP cells, but this repopulation occurred more rapidly in SP cells than non-SP. An MTT assay and cell cycle distribution analysis reveal a similar profile between SP and non-SP groups. However, in the presence of doxorubicin (DOX) and methotrexate (MTX), SP cells showed significantly lower Annexin V staining when compared to non-SP cells. Taken together, these results demonstrate that SP cells have an active regeneration capacity and high anti-apoptotic activity compared with non-SP cells. Furthermore, our GeneChip data revealed a heightened mRNA expression of ABCG2 and ABCC2 in SP cells. Overall these data explain why the SP of A549 has a unique ability to resist DOX and MTX treatments. Therefore, we suggest that the expression of the ABCG2 transporter plays an important role in the multidrug resistance phenotype of A549 SP cells.

Interferon-gamma regulates idiopathic pneumonia syndrome, a Th17+CD4+ T-cell-mediated graft-versus-host disease

RATIONALE: Pulmonary complications of hematopoietic stem cell transplantation include infections and graft-versus-host diseases, such as idiopathic pneumonia syndrome (IPS). Conflicting data exist regarding the role of the interferon (IFN)-gamma-producing Th1 CD4(+) T-cell subset and IL-17A in IPS. OBJECTIVES: To determine the role of IFN-gamma and IL-17A in the establishment of pulmonary graft-versus-host disease. METHODS: A semiallogeneic murine model based on C57BL/6 x BALB/c as recipients with transplantation of BALB/c RAG2(-/-) bone marrow and transfer of different genetic knockout T cells (T-bet(-/-), IFN-gamma(-/-), IFN-gammaR(-/-)) on a BALB/c background. Lung tissue was examined for parenchymal changes and infiltrating cells by histology and fluorescence-activated cell sorter analysis. MEASUREMENTS AND MAIN RESULTS: After transfer of semiallogeneic bone marrow together with donor CD4(+) T cells lacking IFN-gamma or T-bet-a T-box transcription factor controlling Th1 commitment-we found severe inflammation in the lungs, but no enhancement in other organs. In contrast, wild-type donor CD4(+) T cells mediated minimal inflammation only, and donor CD8(+) T cells were not required for IPS development. Mechanistically, the absence of IFN-gamma or IFN-gamma signaling in pulmonary parenchymal cells promoted expansion of IL-17A-producing CD4(+) T cells and local IL-17A release. In vivo depletion of IL-17A reduced disease severity. CONCLUSIONS: One mechanism of IFN-gamma protection against IPS is negative regulation of the expansion of pathogenic IL-17A-producing CD4(+) T cells through interaction with the IFN-gamma receptor on the pulmonary parenchymal cell population.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

FUNCTIONALIZATION OF CARBON NANOTUBES FOR MESENCHYMAL STEM CELL-ASSISTED PHOTOTHERMAL THERAPY

Carbon nanotubes were first cut and functionalized with a newly developed reaction involving autoclaving and sonication in hydrogen peroxide. The functionalized nanotubes were characterized and evaluated for aqueous solubility. Studies which relate reaction conditions to final carbon nanotube length were conducted. Hydroxyl groups present on the carbon nanotubes served as a platform for a series of addition reactions, with the objective of conjugating streptavidin and fluorescent markers onto the carbon nanotubes. The modified nanotubes were attached onto the surface of biotinylated mesenchymal stem cells, creating a novel, tumor-homing delivery system for photothermal anticancer agents.

A 5-year longitudinal study of the relationships between stress, coping, and immune cell beta(2)-adrenergic receptor sensitivity

Caring for a spouse with Alzheimer's disease (AD) is associated with overall health decline and impaired cardiovascular functioning. This morbidity may be related to the effects of caregiving stress and impaired coping on beta(2)-adrenergic receptors, which mediate hemodynamic and vascular responses and are important for peripheral blood mononuclear cell (PBMC) trafficking and cytokine production. This study investigated the longitudinal relationship between stress, personal mastery, and beta(2)-adrenergic receptor sensitivity assessed in vitro on PBMC. Over a 5-year study, 115 spousal AD caregivers completed annual assessments of caregiving stress, mastery, and PBMC beta(2)-adrenergic receptor sensitivity, as assessed by in vitro isoproterenol stimulation. Heightened caregiving stress was associated with significantly decreased receptor sensitivity, whereas greater sense of personal mastery was associated with significantly increased receptor sensitivity. These results suggest that increased stress may be associated with a desensitization of beta(2)-receptors, which may contribute to the development of illness among caregivers. However, increased mastery is associated with increased receptor sensitivity, and may therefore serve as a resource factor for improved health in this population.

Tenascin-C and tenascin-W in whisker follicle stem cell niches: possible roles in regulating stem cell proliferation and migration

The whisker follicle has CD34-positive stem cells that migrate from their niche near the bulge along the glassy membrane to the whisker bulb, where they participate in the formation of the whisker shaft. Using immunohistochemistry we found the glycoprotein tenascin-C in the fibrous capsule of mouse whisker follicles, along the glassy membrane and in the trabecular region surrounding keratin-15-negative, CD34-positive stem cells. The related glycoprotein tenascin-W is found in the CD34-positive stem cell niche, in nearby trabeculae, and along the glassy membrane. Tenascin-W is also found in the neural stem cell niche of nearby hair follicles. The formation of stress fibers and focal adhesion complexes in CD34-positive whisker-derived stem cells cultured on fibronectin was inhibited by both tenascin-C and tenascin-W, which is consistent with a role for these glycoproteins in promoting the migration of these cells from the niche to the whisker bulb. Tenascin-C, but not tenascin-W, increased the proliferation of whisker follicle stem cells in vitro. Thus, the CD34-positive whisker follicle stem cell niche contains both tenascin-C and tenascin-W, and these glycoproteins may play a role in directing the migration and proliferation of these stem cells.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

Increased expression of putative cancer stem cell markers in the bone marrow of prostate cancer patients is associated with bone metastasis progression.

BACKGROUND The number of cells positive for the α-6 and α-2 integrin subunits and the c-Met receptor in primary tumors and bone biopsies from prostate cancer patients has been correlated with metastasis and disease progression. The objective of this study was to quantify disseminated tumour cells present in bone marrow in prostate cancer patients using specific markers and determine their correlation with metastasis and survival. METHODS Patients were included at different stage of prostate cancer disease, from localised to metastatic castration-resistant prostate cancer. Healthy men were used as a control group. Bone marrow samples were collected and nucleated cells separated. These were stained for CD45, α-2, α-6 integrin subunits and c-Met and samples were processed for analysis and quantification of CD45-/α2+/α6+/c-met + cells using flow cytometry. Clinical and pathological parameters were assessed and survival measured. Statistical analyses were made of associations between disease specific parameters, bone marrow flow cytometry data, prostate-specific antigen (PSA) progression free survival and bone metastases progression free survival. RESULTS For all markers, the presence of more than 0.1% positive cells in bone marrow aspirates was significantly associated with the risk of biochemical progression, the risk of developing metastasis and death from prostate cancer. CONCLUSIONS Quantification of cells carrying putative stem cell markers in bone marrow is a potential indicator of disease progression. Functional studies on isolated cells are needed to show more specifically their property for metastatic spread in prostate cancer.

Tenascins in stem cell niches.

Tenascins are extracellular matrix proteins with distinct spatial and temporal expression during development, tissue homeostasis and disease. Based on their expression patterns and knockout phenotypes an important role of tenascins in tissue formation, cell adhesion modulation, regulation of proliferation and differentiation has been demonstrated. All of these features are of importance in stem cell niches where a precise regulation of growth versus differentiation has to be guaranteed. In this review we summarize the expression and possible functions of tenascins in neural, epithelial and osteogenic stem cell niches during normal development and organ turnover, in the hematopoietic and pro-inflammatory niche as well as in the metastatic niche during cancer progression.

In vitro cell motility as a potential mesenchymal stem cell marker for multipotency.

Mesenchymal stem cells (MSCs) are expected to have a fundamental role in future cell-based therapies because of their high proliferative ability, multilineage potential, and immunomodulatory properties. Autologous transplantations have the "elephant in the room" problem of wide donor variability, reflected by variability in MSC quality and characteristics, leading to uncertain outcomes in the use of these cells. We propose life imaging as a tool to characterize populations of human MSCs. Bone marrow MSCs from various donors and in vitro passages were evaluated for their in vitro motility, and the distances were correlated to the adipogenic, chondrogenic, and osteogenic differentiation potentials and the levels of senescence and cell size. Using life-image measuring of track lengths of 70 cells per population for a period of 24 hours, we observed that slow-moving cells had the higher proportion of senescent cells compared with fast ones. Larger cells moved less than smaller ones, and spindle-shaped cells had an average speed. Both fast cells and slow cells were characterized by a low differentiation potential, and average-moving cells were more effective in undergoing all three lineage differentiations. Furthermore, heterogeneity in single cell motility within a population correlated with the average-moving cells, and fast- and slow-moving cells tended toward homogeneity (i.e., a monotonous moving pattern). In conclusion, in vitro cell motility might be a useful tool to quickly characterize and distinguish the MSC population's differentiation potential before additional use.