Vaughan Feisst

Concise review: human adipose-derived stem cells: separating promise from clinical need

Human adipose‐derived stem cells (ASCs) have become an increasing interest to both stem cell biologists and clinicians because of their potential to differentiate into adipogenic, osteogenic, chondrogenic, and other mesenchymal lineages, as well as other clinically useful properties attributed to them, such as stimulation of angiogenesis and suppression of inflammation. ASCs have already been used in a number of clinical trials, and some successful outcomes have been reported, especially in tissue reconstruction. However, a critical review of the literature reveals considerable uncertainty about the true clinical potential of human ASC. First, the surgical needs that ASC might answer remain relatively few, given the current difficulties in scaling up ASC‐based tissue engineering to a clinically useful volume. Second, the differentiation of ASC into cell lineages apart from adipocytes has not been conclusively demonstrated in many studies due to the use of rather simplistic approaches to the confirmation of differentiation, such as the use of nonspecific histological dyes, or a small number of molecular markers of uncertain significance. Third, the ASC prepared from human lipoaspirate for different studies differ in purity and molecular phenotype, with many studies using cell preparations that are likely to contain heterogeneous populations of cells, making it uncertain whether ASC themselves are responsible for effects observed. Hence, while one clinical application already looks convincing, the full clinical potential of ASC awaits much deeper investigation of their fundamental biology. STEM CELLS 2011,29:404–411

From bench to bedside: use of human adipose-derived stem cells.

Since the discovery of adipose-derived stem cells (ASC) in human adipose tissue nearly 15 years ago, significant advances have been made in progressing this promising cell therapy tool from the laboratory bench to bedside usage. Standardization of nomenclature around the different cell types used is finally being adopted, which facilitates comparison of results between research groups. In vitro studies have assessed the ability of ASC to undergo mesenchymal differentiation as well as differentiation along alternate lineages (transdifferentiation). Recently, focus has shifted to the immune modulatory and paracrine effects of transplanted ASC, with growing interest in the ASC secretome as a source of clinical effect. Bedside use of ASC is advancing alongside basic research. An increasing number of safety-focused Phase I and Phase IIb trials have been published without identifying any significant risks or adverse events in the short term. Phase III trials to assess efficacy are currently underway. In many countries, regulatory frameworks are being developed to monitor their use and assure their safety. As many trials rely on ASC injected at a distant site from the area of clinical need, strategies to improve the homing and efficacy of transplanted cells are also being explored. This review highlights each of these aspects of the bench-to-bedside use of ASC and summarizes their clinical utility across a variety of medical specialties.

Cultured pericytes from human brain show phenotypic and functional differences associated with differential CD90 expression

The human brain is a highly vascular organ in which the blood-brain barrier (BBB) tightly regulates molecules entering the brain. Pericytes are an integral cell type of the BBB, regulating vascular integrity, neuroinflammation, angiogenesis and wound repair. Despite their importance, identifying pericytes amongst other perivascular cell types and deciphering their specific role in the neurovasculature remains a challenge. Using primary adult human brain cultures and fluorescent-activated cell sorting, we identified two CD73+CD45− mesenchymal populations that showed either high or low CD90 expression. CD90 is known to be present on neurons in the brain and peripheral blood vessels. We found in the human brain, that CD90 immunostaining localised to the neurovasculature and often associated with pericytes. In vitro, CD90+ cells exhibited higher basal proliferation, lower expression of markers αSMA and CD140b, produced less extracellular matrix (ECM) proteins, and exhibited lesser pro-inflammatory responses when compared to the CD90− population. Thus, CD90 distinguishes two interrelated, yet functionally distinct pericyte populations in the adult human brain that may have discrete roles in neurovascular function, immune response and scar formation.

MicroRNA regulation in human CD8+ T cell subsets--cytokine exposure alone drives miR-146a expression.

BackgroundmicroRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation. We also investigated how changes in miR-146a related to other miRNAs that alter with CD8+ differentiation status.MethodsWe treated human CD8+ T cells with the cytokines IL-2, IL-7 or IL-15 either at rest or after stimulation with anti-CD3 and anti-CD28. For some experiments we also purified human CD8+ T cell subsets ex vivo. Flow cytometry was used in parallel to assess cell surface memory marker expression. Total RNA from these cells was subjected to microarray analysis and real-time PCR for miRNA expression. Nucleofection studies were performed to assess potential mRNA targets of miR-146a.ResultsWe find that miR-146a is up-regulated in naïve CD8+ T cells exposed to IL-2 or IL-15, even in the absence of an activating T cell receptor stimulus, but not when IL-7 is also present. miR-146a expression correlates with a memory phenotype in both ex vivo and in vitro cultured cells although in our hands overexpression of miR-146a was not sufficient alone to drive a full memory phenotype. In ex vivo analysis, miR-146a was one of a small number of miRNAs that was differentially expressed between naïve and memory CD8+ T cells.ConclusionsmiR-146a is emerging as a critical regulator of immune system. Our data shows that miR-146a expression is strongly influenced by the cytokine milieu even in the absence of a T cell receptor stimulus. Our results have implications for studies designed to assess the function of miR-146a, help to define a fingerprint of miRNA expression in CD8+ T cell subsets and may be useful when designing optimal protocols for T cell expansion as efficacy of T cell immunotherapy is correlated with an `early’ memory phenotype.

Ahnak is downregulated in melanoma, predicts poor outcome, and may be required for the expression of functional cadherin-1

The aim of this study was to further our understanding of the transformation process by identifying differentially expressed proteins in melanocytes compared with melanoma cell lines. Tandem mass spectrometry incorporating iTRAQ reagents was used as a screen to identify and comparatively quantify the expression of proteins in membrane-enriched samples isolated from primary human melanocytes or three melanoma cells lines. Real-time PCR was used to validate significant hits. Immunohistochemistry was used to validate the expression of proteins of interest in melanocytes in human skin and in melanoma-infiltrated lymph nodes. Publically available databases were examined to assess mRNA expression and correlation to patient outcome in a larger cohort of samples. Finally, preliminary functional studies were carried out using siRNAs to reduce the expression of a protein of interest in primary melanocytes and in a keratinocyte cell line. Two proteins, AHNAK and ANXA2, were significantly downregulated in the melanoma cell lines compared with melanocytes. Downregulation was confirmed in tumor cells in a subset of human melanoma-infiltrated human lymph nodes compared with melanocytes in human skin. Examination of Gene Expression Omnibus database data sets suggests that downregulation of AHNAK mRNA and mutation of the AHNAK gene are common in metastatic melanoma and correlates to a poor outcome. Knockdown of AHNAK in primary melanocytes and in a keratinocyte cell line led to a reduction in detectable cadherin-1. This is the first report that we are aware of which correlates a loss of AHNAK with melanoma and poor patient outcome. We hypothesize that AHNAK is required for the expression of functional cadherin-1.

Modulation of cell adhesion to conductive polymers

Our work on conductive polymer (CP) systems grafted with stimuli-responsive polymer brushes is motivated by the prospect of precisely controlling cellular behaviour by tailored smart interfaces. Here, the effects on cell adhesion by applying a potential to poly(3,4-ethylenedioxythiophene) (PEDOT) during both protein coating and cell culture is investigated. The results highlight the importance of pre-adsorbing fibronectin in this case, especially for the reduced polymer which binds protein strongly. The effects of changing the surface chemistry of the PEDOT electrode by grafting of brushes by atom transfer radical polymerisation (ATRP) is also investigated. Specifically, the composition of the salt-sensitive poly(oligo(ethylene glycol methyl ether methacrylate))-based brushes was tailored to control the level of cell adhesion to the interface. The composition, and also the length of the grafted brushes was seen to be important to the cell adhesion. It is also demonstrated how PEDOT films grafted with a protein and cell rejecting brush can be converted to a cell adhesive state by attaching an integrin ligand to the brush to mediate cell adhesion.

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Individual and cocktails of TLR ligands influence APCs in situ in human skin explants

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.Changes in microbiome, mucosal immunity and intestinal integrity have been associated with the onset of Type 1 Diabetes (T1D) in children. Toll-like Receptors (TLR) have been associated all three factors. The role of TLR and their effects on microbiome in autoimmunity were studied by crossing TLR1,2,4,6,9 and MyD88 targeted deficiency mutations to the type 1 diabetes (T1D)-prone NOD mouse strain. While NOD.Tlr9-/- and NOD.Tlr6-/- mice were unaffected, T1D in NOD.Tlr4-/- and NOD.Tlr1-/- mice was exacerbated and that in NOD.Myd88-/- and NOD.Tlr2-/- mice ameliorated. Physical parameters of the intestines were compared; ileal weight was reduced in NOD.Tlr1-/-mice. Similarly, by histology, these mice had reduced villus length and width. The intestinal microbiomes of NOD wild-type (WT), NOD.Tlr1-/-, NOD.Tlr2-/- and NOD.Tlr4-/- mice were compared by high throughput sequencing of 16S ribosomal DNA (rDNA), in two cohorts, 18 months apart. Analysis of caecal 16S sequences clearly resolved the mouse lines and there were significant differences in beta diversity between the strains, with individual bacterial species contributing greatly to the differences in the microbiota of individual TLR-deficient strains. To test the relationship between microbiome and T1D, all strains were re-derived onto the parental NOD/Lt line and the incidence of T1D re-assessed within two generations. All rederived lines expressed an incidence of disease similar to that of the parental line. TLR deficiencies are associated with changes in microbiome; changes of microbiome are associated with T1D; the effects of TLR deficiencies on T1D appear to be mediated by their effects on gut flora.Intestinal TCRb+CD4-CD8b-CD8a+ (CD8aa) IELs alleviate T cell induced colitis and have been suggested to play a role in virus infection and cancer. Their thymic development has been elucidated to some extent, as IEL precursors (IELp) are known to be CD4-CD8-CD5+TCRb+, but is not yet fully understood. Within the thymus, mature IELp were identified based on their expression of CD122 and MHC class I. Two major phenotypic subsets exist within this mature thymic IELp population: a PD1+Tbet- population that preferentially expresses a4b7, and a PD1-Tbet+ population with preferential CD103 expression. These two populations were also distinct in their Valpha repertoire. The PD1+a4b7+ population contains clones that are strongly self-reactive as judged by Nur77GFP and their dramatic increase in Bim deficient mice, while the PD1-Tbet+ population did not show these characteristics. Both gave rise to CD8aa IELs upon adoptive transfer into RAG-/- recipients. However intrathymic labeling revealed that PD1+a4b7+ IELp were the major thymic emigrating population, and emigration was S1P1-dependent. In contrast, PD1-Tbet+ IELp expressed CXCR3, were retained, and accumulated in the thymus with age. Preliminary immunofluorescence data furthermore indicate differential thymic cortico-medullary localization of the IELp subtypes. These experiments more precisely define the behavior of IEL precursors.

Adipose-Derived Stem Cells: Isolation and Culturing

The use of human adipose-derived stem cells (ASC) for research and clinical purposes has become increasingly common over the past years. The advantages of ASC over more traditional sources of adult stem cells such as bone marrow (BM) include it’s ready availability in most people, the ease of significant volume fat removal by liposuction and subsequently the large number of adult mesenchymal stem cells (MSC) able to be isolated from each harvest. This chapter provides clear, focused methods for the isolation of the stromal vascular fraction (SVF) from lipoaspirate, which contains the ASC. Methods for subsequent culture, passage and cryopreservation of ASC are given, to enable other researchers to work with this exciting resource.

Human Adipose-Derived Stem Cells (ASC): Their efficacy in clinical applications

Mesenchymal stem cells (MSC) have been used for therapeutic purposes for many years. Multipotent cells in adipose tissue were postulated to exist by Kaplan and colleagues during their investigations into a disease called progressive osseous heteroplasia (POH). In this disease, bone forms in atypical locations, such as in subcutaneous fat or in muscle. Interest in multilineage cells from adipose tissue gained momentum after 2001, when Zuk and her colleagues published their sentinel paper showing differentiation of stromal-type cells from adipose tissue along adipogenic, chondrogenic and osteogenic lineages. Such has been the explosion of interest in the topic, a Medline search with “adipose” and “stem” and “cell” as the key words returns nearly 3,000 articles published since this time, compared with only 300 in the 30 years prior to this. The cells isolated from adipose tissue have been given various names, including adipose stem cells, adipose-derived stem cells, adipose-derived stromal cells, among others. A consensus statement was published following the International Fat Applied Technology Society (IFATS, now known as the International Federation for Adipose Therapeutics and Science) 2nd international meeting in 2004, concluding that they should be referred to as adipose-derived stem/stromal cells (ASC) to promote consistency across research group; hence, this is the terminology used in this article.