Immunodepletion and hypoxia preconditioning of mouse vompact bone cells as a novel protocol to isolate highly immunosuppressive mesenchymal stem cells

Prepublished on Liebert Instant Online December 21, 2016Compact bones (CB) are major reservoirs of mouse mesenchymal stem cells (mMSC). Here, we established a protocol to isolate MSC from CB and tested their immunosuppressive potential. Collagenase type II digestion of BM-flushed CB from C57B/6 mice was performed to liberate mMSC precursors from bone surfaces to establish nondepleted mMSC. CB cells were also immunodepleted based on the expression of CD45 (leukocytes) and TER119 (erythroid cells) to eliminate hematopoietic cells. CD45-TER119- CB cells were subsequently used to generate depleted mMSC. CB nondepleted and depleted mMSC progenitors were cultured under hypoxic conditions to establish primary mMSC cultures. CB depleted mMSC compared to nondepleted mMSC showed greater cell numbers at subculturing and had increased functional ability to differentiate into adipocytes and osteoblasts. CB depleted mMSC had high purity and expressed key mMSC markers (>85% Sca-1, CD29, CD90) with no mature hematopoietic contaminating cells (<5% CD45, CD11b) when subcultured to passage 5 (P5). Nondepleted mMSC cultures, however, were less pure and heterogenous with <72% Sca-1+, CD29+, and CD90+ cells at early passages (P1 or P2), along with high percentages of contaminating CD11b+ (35.6%) and CD45+ (39.2%) cells that persisted in culture long term. Depleted and nondepleted mMSC nevertheless exhibited similar potency to suppress total (CD3+), CD4+ and CD8+ T cell proliferation, in a dendritic cell allostimulatory one-way mixed lymphocyte reaction. CB depleted mMSC, pretreated with proinflammatory cytokines IFN-γ, TNF-α, and IL-17A, showed superior suppression of CD8+ T cell, but not CD4+ T cell proliferation, relative to untreated-mMSC. In conclusion, CB depleted mMSC established under hypoxic conditions and treated with selective cytokines represent a novel source of potent immunosuppressive MSC. As these cells have enhanced immune modulatory function, they may represent a superior product for use in clinical allotransplantation.Kisha Nandini Sivanathan, Stan Gronthos, Shane T. Grey, Darling Rojas-Canales, and Patrick T. Coate

Transcriptome profiling of IL-17A preactivated mesenchymal stem cells: a comparative study to unmodified and IFN-gamma modified mesenchymal stem cells

Published 15 February 2017Human mesenchymal stem cells pretreatment with IL-17A (MSC-17) potently enhances T cell immunosuppression but not their immunogenicity, in addition to avidly promoting the induction of suppressive regulatory T cells. The aim of this study was to identify potential mechanisms by which human MSC-17 mediate their superior immunomodulatory function. Untreated-MSC (UT-MSC), IFN-γ treated MSC (MSC-γ), and MSC-17 were assessed for their gene expression profile by microarray. Significantly regulated genes were identified for their biological functions (Database for Annotation, Visualisation and Integrated Discovery, DAVID). Microarray analyses identified 1278 differentially regulated genes between MSC-γ and UT-MSC and 67 genes between MSC-17 and UT-MSC. MSC-γ were enriched for genes involved in immune response, antigen processing and presentation, humoral response, and complement activation, consistent with increased MSC-γ immunogenicity. MSC-17 genes were associated with chemotaxis response, which may be involved in T cell recruitment for MSC-17 immunosuppression. MMP1, MMP13, and CXCL6 were highly and specifically expressed in MSC-17, which was further validated by real-time PCR. Thus, MMPs and chemokines may play a key role in mediating MSC-17 superior immunomodulatory function. MSC-17 represent a potential cellular therapy to suppress immunological T cell responses mediated by expression of an array of immunoregulatory molecules.Kisha Nandini Sivanathan, Darling Rojas-Canales, Shane T. Grey, Stan Gronthos, and Patrick T. Coate

Cellular therapy for cardiovascular disease Part 2 - Delivery of cells and clinical experience

Peter J Psaltis, Stan Gronthos, Stephen G Worthley and Andrew C.W. Zannettin

Immunomodulatory properties of induced pluripotent stem cell-derived mesenchymal cells

Abstract not availableJia Ng, Kim Hynes, Gregory White, Kisha Nandini Sivanathan, Kate Vandyke, Peter Mark Bartold and Stan Grontho

Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow

Previous studies have provided evidence for the existence of adult human bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells. Using a combination of cell separation techniques, we have isolated an almost homogeneous population of BMSSCs from adult human bone marrow. Lacking phenotypic characteristics of leukocytes and mature stromal elements, BMSSCs are non-cycling and constitutively express telomerase activity in vivo. This mesenchymal stem cell population demonstrates extensive proliferation and retains the capacity for differentiation into bone, cartilage and adipose tissue in vitro. In addition, clonal analysis demonstrated that individual BMSSC colonies exhibit a differential capacity to form new bone in vivo. These data are consistent with the existence of a second population of bone marrow stem cells in addition to those for the hematopoietic system. Our novel selection protocol provides a means to generate purified populations of BMSSCs for use in a range of different tissue engineering and gene therapy strategies.Stan Gronthos, Andrew C. W. Zannettino, Shelley J. Hay, Songtao Shi, Stephen E. Graves, Angela Kortesidis and Paul J. Simmon

Insulin-like Growth Factor 1 and Transforming Growth Factor-β Stimulate Cystine/Glutamate Exchange Activity in Dental Pulp Cells

Introduction The growth factors insulin-like growth factor (IGF-1) and transforming growth factor-β (TGF-β) are protective to dental pulp cells in culture against the toxicity of the composite materials Durafill VS and Flow Line (Henry Schein Inc, New York, NY). Because the toxicity of these materials is mediated by oxidative stress, it seemed possible that the protective effects of IGF-1 and TGF-β were through the enhancement of an endogenous antioxidant mechanism. Methods We used cultured dental pulp cells to determine the mechanism of the protective effects of IGF-1 and TGF-β, focusing on the glutathione system and the role of cystine/glutamate exchange (system xc-). Results We found that the toxicity of Durafill VS and Flow Line was attenuated by the addition of glutathione monoethylester, suggesting a specific role for the cellular antioxidant glutathione. Supporting this hypothesis, we found that IGF-1 and TGF-β were protective against the toxicity of the glutathione synthesis inhibitor buthionine sulfoximine. Because levels of cellular cystine are the limiting factor in the production of glutathione, we tested the effects of IGF-1 and TGF-β on cystine uptake. Both growth factors stimulated system xc–mediated cystine uptake. Furthermore, they attenuated the glutathione depletion induced by Durafill VS and Flow Line. Conclusions The results suggest that IGF-1 and TGF-β are protective through the stimulation of system xc–mediated cystine uptake, leading to maintenance of cellular glutathione. This novel action of growth factors on dental pulp cells has implications not only for preventing toxicity of dental materials but also for the general function of these cells

The Role of Clinical Features in the Diagnostic Reasoning of Psychologists

This item is only available electronically.Across health and medical domains, experts rely on idiosyncratic case-based pattern recognition to rapidly and accurately identify significant features that define a case. Understanding how clinical psychologists use features to form a diagnosis can provide valuable insights into changes in diagnostic performance as a function of experience. Previous studies examining the diagnostic accuracy of Clinical Psychologists have demonstrated that practicing Clinical Psychologists are no more accurate at diagnosing mental health conditions than Undergraduate psychology students. This study aimed to explore how the interpretation and use of clinical features develops with experience to facilitate diagnostic reasoning. Undergraduate psychology students (n = 24), Clinical Masters students (n = 2) and Clinical Psychologists (n = 10) were presented with eight mental health case studies. The case studies contained a combination of seven features: those shared between the possible diagnoses and those unique to the primary diagnosis and contextual features. Participants were prompted to give primary and secondary diagnoses for the case studies then asked to rate the extent to which each of the seven features supported the primary and secondary diagnoses. On average, Clinical Psychologists displayed the best diagnostic accuracy. Additionally, tertiary education predicted diagnostic accuracy and the use of unique features but clinical experience was predictive of neither. Rather, clinical experience predicted the use of contextual features (i.e. the character’s age or occupation). Future research should extend on these findings using real-life case studies and non-aggregated feature acquisition data.Thesis (B.PsychSc(Hons)) -- University of Adelaide, School of Psychology, 202

Characterization of bone marrow derived mesenchymal stem cells in suspension

INTRODUCTION: Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs). METHODS: To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice. RESULTS: S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production. CONCLUSIONS: These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs

A novel method for the isolation of subpopulations of rat adipose stem cells with different proliferation and osteogenic differentiation potentials

Bone marrow has been the elected cell source of studies published so far concerning bone and cartilage tissue-engineering approaches. Recent studies indicate that adipose tissue presents significant advantages over bone marrow as a cell source for tissue engineering. Most of these studies report the use of adipose stem cells (ASCs) isolated by a method based on the enzymatic digestion of the adipose tissue and on the ability of stem cells to adhere to a cell culture plastic surface. Using this method, a heterogeneous population was obtained containing different cell types that have been shown to compromise the proliferation and differentiation potential of the stem cells. This paper reports the development and optimization of a new isolation method that enables purified cell populations to be obtained that exhibit higher osteogenic differentiation and/or proliferation potential. This method is based on the use of immunomagnetic beads coated with specific antibodies and it is compared with other methods described in the literature for the selection of stem cell populations, e.g. methods based on a gradient solution and enzymatic digestion. The results showed that the isolation method based on immunomagnetic beads allows distinct subpopulations of rat ASCs to be isolated, showing different stem cells marker expressions and different osteogenic differentiation potentials. Therefore, this method can be used to study niches in ASC populations and/or also allow adipose tissue to be used as a stem cell source in a more efficient manner, increasing the potential of this cell source in future clinical applications.T. Rada thanks the EU Marie Curie Actions Alea Jacta Est for a PhD fellowship. This work was partially supported by the European Union-funded STREP Project HIPPOCRATES (Grant No. NMP3-CT-2003-505758) and was carried out under the scope of the European NoE EXPERTISSUES (Grant No. NMP3-CT-2004-500283)