Detailed immunophenotyping of the hematopoietic graft from patients with multiple sclerosis undergoing autologous hematopoietic stem cell transplant.

BACKGROUND AIMS: Autologous hematopoietic stem cell transplantation (AHSCT) is a highly effective therapy for relapsing multiple sclerosis. Re-infused stem cells provide "rescue" from the pancytopenia induced by immuno-chemotherapy. To date, no study has analyzed the non-stem cell content of the leukapheresis product (graft) in regards to its influence on disease remission in AHSCT for multiple sclerosis (MS). METHODS: Detailed immunophenotyping of the stem cell graft was performed in a cohort of highly active patients with MS (n = 22) followed for a median of 6 years' post-AHSCT. RESULTS: Effector memory populations thought to house pathogenic clones including Th17 cells and central nervous system homing T cells were detected in the graft at similar proportions to pre-AHSCT. There was no association between absolute counts of these populations in the graft and treatment response. Only in responder patients was there evidence of a significant decrease in these putative pro-inflammatory populations by 3 months' post-transplant. Although there was no statistical difference in the number of T regulatory cells (Tregs) in the graft between responders and relapsing patients, the absolute count of Tregs in the graft correlated with circulating Tregs in the first 6 months post-AHSCT in responders alone. CONCLUSIONS: Our results collectively suggest that the early establishment of immune tolerance post-AHSCT appears to relate to a decrease in putative pathogenic cell populations following reinfusion, and that Treg load in the leukapheresis product is less relevant to treatment response than the early expansion of graft-derived Tregs. It therefore remains unclear whether employment of CD34 selection to manipulate the graft may offer additional benefit in remission rates post-AHSCT for MS. Cellular therapy targeted toward early Treg expansion may provide recourse for long-term remission rates in MS.

Diversification and expansion of the EBV-reactive cytotoxic T lymphocyte repertoire following autologous haematopoietic stem cell transplant for multiple sclerosis.

Both genetic susceptibility and environmental exposures are thought to be involved in multiple sclerosis (MS) pathogenesis. Of all viruses potentially relevant to MS aetiology, Epstein-Barr virus (EBV) is the best-studied. EBV is a B cell lymphotropic virus which is able to evade the immune system by establishing latent infection in memory B cells, and EBV reactivation is restricted by CD8 cytotoxic T cell (CTL) responses in immune competent individuals. Autologous haematopoietic stem cell transplantation (AHSCT) is considered to be the most effective therapy in the treatment of relapsing MS even though chemotherapy-induced lymphopenia can associate with the re-emergence of latent viruses. Despite the increasing interest in EBV and MS pathogenesis the relationship between AHSCT, EBV and viral immunity in people with MS has not been investigated to date. This study analysed immune responses to EBV in a well characterised cohort of 13 individuals with MS by utilising pre-AHSCT, and 6-, 12- and 24-month post AHSCT bio-banked peripheral blood mononuclear cells and plasma samples. It is demonstrated that the infused stem cell product contains latently EBV-infected memory B cells, and that EBV viremia occurs in the immune-compromised recipient post-transplant. High throughput TCR analysis detected expansion and diversification of the CD8 CTL responses reactive with EBV lytic and latent antigens from 6 to 24 months following AHSCT. Increased levels of latent EBV infection found within the B cell pool following treatment, as measured by EBV genomic detection, did not associate with disease relapse. This is the first study of EBV immunity following application of AHSCT in the treatment of MS and not only raises important questions about the role of EBV infection in MS pathogenesis, but is of clinical importance given the expanding clinical trials of adoptive EBV-specific CTLs in MS.

Isolation and Characterisation of an Adipose-derived human mesenchymal stem cell line - 'CKC-Endeavour-1'.

Mesenchymal stem cells derived from adipose tissue (ADMSCs) are being increasingly considered in regenerative medicine-based clinical applications. Apart from possessing therapeutic applications themselves, ADMSCs also secrete a myriad of soluble factors which are promising candidates for treating several degenerative diseases such as osteoarthritis and neurodegenerative diseases, wound repair as well as for cosmeceutical purposes. In our research study, we successfully isolated ADMSCs in-house, now called CKC-Endeavour-1 from the lipoaspirate sample of a patient who underwent liposuction. The subsequent expansion of cells was performed in xeno-free and serum-free conditions and their characterisation was performed using tri-lineage differentiation studies. The levels of differentiation were assessed by staining and gene expression which was observed to be comparable between the in-house developed ADMSC cell line and the commercially purchased ADMSCs. Following characterisation, the secretory components from these MSCs, namely, conditioned media (ADMSC-CM) and exosomes (ADMSC-EXO) were harvested from CKC-Endeavour-1 under xeno-free, serum-free, and supplement-free conditions followed by lyophilisation in order to attempt to prolong its shelf-life. The comprehensive analysis of the secretome profile of ADMSC-CM using carried out using cytokine array and demonstrated the presence of 105 cytokines and growth factors. Also, clinical grade Izon columns were used to isolate the exosomes from ADMSC-CM obtaining exosomes in the size range of <200nm, analysed using nanoparticle tracking analysis. Overall, our study developed an ADMSC cell line, CKC-Endeavour-1, along with their CM and exosome (EXO) products under clinically safe conditions. Additionally, we have obtained a comprehensive understanding of the secreted factors present in the ADMSC-CM which could be further explored in detail to tap the best therapeutic benefits from them.

Sustained immunotolerance in multiple sclerosis after stem cell transplant.

OBJECTIVE: Autologous haematopoietic stem cell transplantation (AHSCT) has the potential to induce sustained periods of disease remission in multiple sclerosis (MS), which is an inflammatory disease of the central nervous system (CNS) characterised by demyelination and axonal degeneration. However, the mechanisms associated with durable treatment responses in MS require further elucidation. METHODS: To characterise the longer term immune reconstitution effects of AHSCT at 24 and 36 months (M) post-transplant, high-dimensional immunophenotyping of peripheral blood mononuclear cells from 22 MS patients was performed using two custom-designed 18-colour flow cytometry panels. RESULTS: The higher baseline frequencies of specific pro-inflammatory immune cells (T-helper-17 (Th17) cells, mucosal-associated invariant T-cells and CNS-homing T-conventional (T-conv) cells observed in MS patients were decreased post-AHSCT by 36M. This was accompanied by a post-AHSCT increase in frequencies and absolute counts of immunoregulatory CD56hi natural killer cells at 24M and terminally differentiated CD8+ CD28- CD57+ cells until 36M. A sustained increase in the proportion of naïve B-cells, with persistent depletion of memory B-cells and plasmablasts was observed until 36M. Reconstitution of the B-cell repertoire was accompanied by a reduction in the frequency of circulating T-follicular helper cells (cTfh) expressing programmed cell death-1 (PD1+ ) at 36M. Associations between frequency dynamics and clinical outcomes indicated only responder patients to exhibit a decrease in Th17, CNS-homing T-conv and PD1+ cTfh pro-inflammatory subsets at 36M, and an increase in CD39+ T-regulatory cells at 24M. INTERPRETATION: AHSCT induces substantial recalibration of pro-inflammatory and immunoregulatory components of the immune system of MS patients for up to 36M post-transplant.

Long-Term Suppression of Circulating Proinflammatory Cytokines in Multiple Sclerosis Patients Following Autologous Haematopoietic Stem Cell Transplantation.

Autologous haematopoietic stem cell transplantation (AHSCT) is a therapeutic option for haematological malignancies, such as non-Hodgkin's lymphoma (NHL), and more recently, for autoimmune diseases, such as treatment-refractory multiple sclerosis (MS). The immunological mechanisms underlying remission in MS patients following AHSCT likely involve an anti-inflammatory shift in the milieu of circulating cytokines. We hypothesised that immunological tolerance in MS patients post-AHSCT is reflected by an increase in anti-inflammatory cytokines and a suppression of proinflammatory cytokines in the patient blood. We investigated this hypothesis using a multiplex-ELISA assay to compare the concentrations of secreted cytokine in the peripheral blood of MS patients and NHL patients undergoing AHSCT. In MS patients, we detected significant reductions in proinflammatory T helper (Th)17 cytokines interleukin (IL)-17, IL-23, IL-1ß, and IL-21, and Th1 cytokines interferon (IFN)γ and IL-12p70 in MS patients from day 8 to 24 months post-AHSCT. These changes were not observed in the NHL patients despite similar pre-conditioning treatment for AHSCT. Some proinflammatory cytokines show similar trends in both cohorts, such as IL-8 and tumour necrosis factor (TNF)-α, indicating a probable treatment-related AHSCT response. Anti-inflammatory cytokines (IL-10, IL-4, and IL-2) were only transiently reduced post-AHSCT, with only IL-10 exhibiting a significant surge at day 14 post-AHSCT. MS patients that relapsed post-AHSCT exhibited significantly elevated levels of IL-17 at 12 months post-AHSCT, unlike non-relapse patients which displayed sustained suppression of Th17 cytokines at all post-AHSCT timepoints up to 24 months. These findings suggest that suppression of Th17 cytokines is essential for the induction of long-term remission in MS patients following AHSCT.

Altered Immune Reconstitution in Allogeneic Stem Cell Transplant Recipients With Human Immunodeficiency Virus (HIV).

BACKGROUND: Persons living with human immunodeficiency virus (HIV) are at elevated risk of developing the malignant diseases that require allogeneic stem cell transplantation (ASCT). Recent data suggest that these individuals are also at an elevated risk of certain complications post-ASCT. This risk may result from preexisting HIV-related factors affecting dynamics of immune reconstitution post-ASCT. However, to date, there has been little work describing the dynamics of immune reconstitution post-ASCT in persons with HIV and none comparing these data to controls without HIV. METHODS: We assessed T-cell reconstitution in 6 ASCT with HIV recipients (HIV+ASCT) compared to a control population of 21 ASCT without HIV recipients. In a subset of HIV+ASCT recipients we performed additional flow cytometry profiling of CD8+ T-cell subsets and antigen specificity of reconstituting CD4+ and CD8+ T cells. RESULTS: We observe no difference in post-ASCT CD4+ T cells between HIV+ASCT and HIV-negative ASCT recipients, despite much lower pre-ASCT CD4+ T-cell counts in the HIV+ASCT group. In contrast, we observed significantly higher CD8+ T-cell numbers in the HIV+ASCT group post-ASCT. The reconstituting CD8+ T-cells were predominantly CD45RO+, whereas homing markers and antigen specificity of these cells varied between participants. CONCLUSION: This study represents the most extensive characterization of immune-reconstitution post-ASCT in persons with HIV, and the first to our knowledge to compare these data to ASCT controls without HIV. The results indicate that immune reconstitution in this group can be affected by preexisting HIV infection and post-ASCT antigen exposure.

Aberrant lipid metabolism as an emerging therapeutic strategy to target cancer stem cells.

Emerging evidence in cancer metabolomics has identified reprogrammed metabolic pathways to be a major hallmark of cancer, among which deregulated lipid metabolism is a prominent field receiving increasing attention. Cancer stem cells (CSCs) comprise <0.1% of the tumor bulk and possess high self-renewal, tumor-initiating properties, and are responsible for therapeutic resistance, disease recurrence, and tumor metastasis. Hence, it is imperative to understand the metabolic rewiring occurring in CSCs, especially their lipid metabolism, on which there have been recent reports. CSCs rely highly upon lipid metabolism for maintaining their stemness properties and fulfilling their biomass and energy demands, ultimately leading to cancer growth and invasion. Hence, in this review we will shed light on the aberrant lipid metabolism that CSCs exploit to boost their survival, which comprises upregulation in de novo lipogenesis, lipid droplet synthesis, lipid desaturation, and ß-oxidation. Furthermore, the metabolic regulators involved in the process, such as key lipogenic enzymes, are also highlighted. Finally, we also summarize the therapeutic strategies targeting the key regulators involved in CSCs' lipid metabolism, which thereby demonstrates the potential to develop powerful and novel therapeutics against the CSC lipid metabolome.

Tolerance regeneration by T regulatory cells in autologous haematopoietic stem cell transplantation for autoimmune diseases.

Autologous haematopoietic stem cell transplantation shows increasing promise as a therapeutic option for patients with treatment-refractory autoimmune disease, particularly systemic sclerosis and multiple sclerosis. However, this intensive chemotherapy-based procedure is not always possible due to potential treatment toxicities and comorbidities. The biological mechanisms of how this procedure induces long-term remission in autoimmune disease are increasingly understood. The focus of this review is on recent research findings on the role of CD4+ T regulatory cells (Tregs) in resetting the immune system leading to the eradication of the autoimmune disease after transplantation. Discovery of the precise mechanisms of this process will allow development of novel Treg-based therapies and thus avoid the need for intensive chemotherapy-based treatment for these autoimmune diseases in the future.

The Influence of Breast Tumour-Derived Factors and Wnt Antagonism on the Transformation of Adipose-Derived Mesenchymal Stem Cells into Tumour-Associated Fibroblasts.

Within the tumour stroma, a heterogeneous population of cell types reciprocally regulates cell proliferation, which considerably affects the progression of the disease. In this study, using tumour conditioned medium (TCM) derived from breast tumour cell lines - MCF7 and MDA MB 231, we have demonstrated the differentiation of adipose-derived mesenchymal stem cells (ADSCs) into tumour-associated fibroblasts (TAFs). Since the Wnt signalling pathway is a key signalling pathway driving breast tumour growth, the effect of the Wnt antagonist secreted frizzled-related protein 4 (sFRP4) was also examined. The response of ADSCs to TCM and sFRP4 treatments was determined by using cell viability assay to determine the changes in ADSC viability, immunofluorescence for mesenchymal markers, glucose uptake assay, and glycolysis stress test using the Seahorse Extracellular Flux analyser to determine the glycolytic activity of ADSCs. ADSCs have been shown to acquire a hyper-proliferative state, significantly increasing their number upon short-term and long-term exposure to TCM. Changes have also been observed in the expression of key mesenchymal markers as well as in the metabolic state of ADSCs. SFRP4 significantly inhibited the differentiation of ADSCs into TAFs by reducing cell growth as well as mesenchymal marker expression (cell line-dependent). However, sFRP4 did not induce further significant changes to the altered metabolic phenotype of ADSCs following TCM exposure. Altogether, this study suggests that the breast tumour milieu may transform ADSCs into a tumour-supportive phenotype, which can be altered by Wnt antagonism, but is independent of metabolic changes.

The inhibitory influence of adipose tissue-derived mesenchymal stem cell environment and Wnt antagonism on breast tumour cell lines.

Tumours exhibit a heterogeneous mix of cell types that reciprocally regulate their growth in the tumour stroma, considerably affecting the progression of the disease. Both adipose-derived mesenchymal stem cells and Wnt signalling pathway are vital in driving breast tumour growth. Hence, we examined the effect of secreted factors released by adipose-derived mesenchymal stem cells, and further explored the anti-tumour property of the Wnt antagonist secreted frizzled-related protein 4 (sFRP4) on MCF-7 and MDA-MB-231 breast tumour cells. We observed that conditioned medium and extracellular matrix derived from adipose-derived mesenchymal stem cells inhibited tumour viability. The inhibitory effect of the conditioned medium was retained within its low molecular weight and non-protein component. The conditioned medium also induced apoptosis accompanied by a decrease in the mitochondrial membrane potential in tumour cells, Furthermore, it downregulated the protein expression of active ß-catenin and Cyclin D1, which are major target proteins of the Wnt signalling pathway, and reduced the expression of anti-apoptotic protein Bcl-xL. The combination of conditioned medium and sFRP4 further potentiated the effects, depending on the tumour cell line and experimental assay. We conclude that factors derived from conditioned medium of adipose-derived mesenchymal stem cells and sFRP4 significantly decreased the tumour cell viability and migration rates (MCF-7), accompanied with an enhanced apoptotic activity through inhibition of canonical Wnt signalling. Besides giving an insight to possible paracrine interactions and influence of signalling pathways, reflective of a breast tumour microenvironment, this study emphasises the utilization of cell free-secreted factors and Wnt antagonists to improve conventional anti-cancer strategies.

The Role of Wnt Signalling in Angiogenesis.

Angiogenesis is a normal biological process wherein new blood vessels form from the growth of pre-existing blood vessels. Preventing angiogenesis in solid tumours by targeting pro-angiogenic factors including vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), basic fibroblast growth factor (bFGF), hepatocyte growth factor, and platelet-derived growth factor (PDGF) is currently under investigation for cancer treatment. Concurrently targeting the cell signalling pathways involved in the transcriptional and post-translational regulation of these factors may provide positive therapeutic results. One such pathway is the Wnt signalling pathway. Wnt was first discovered in mice infected with mouse mammary tumour virus, and has been crucial in improving our understanding of oncogenesis and development. In this review, we summarise molecular and cellular aspects of the importance of Wnt signalling to angiogenesis, including ß-catenin-dependent mechanisms of angiogenic promotion, as well as the study of Wnt antagonists, such as the secreted frizzled-related protein family (SFRPs) which have been shown to inhibit angiogenesis. The growing understanding of the underlying complexity of the biochemical pathways mediating angiogenesis is critical to the identification of new molecular targets for therapeutic applications.

Multi-lineage differentiation of mesenchymal stem cells - To Wnt, or not Wnt.

Mesenchymal stem cells (MSCs) are multipotent precursor cells originating from several adult connective tissues. MSCs possess the ability to self-renew and differentiate into several lineages, and are recognized by the expression of unique cell surface markers. Several lines of evidence suggest that various signal transduction pathways and their interplay regulate MSC differentiation. To that end, a critical player in regulating MSC differentiation is a group of proteins encoded by the Wnt gene family, which was previously known for influencing various stages of embryonic development and cell fate determination. As MSCs have gained significant clinical attention for their potential applications in regenerative medicine, it is imperative to unravel the mechanisms by which molecular regulators control differentiation of MSCs for designing cell-based therapeutics. It is rather coincidental that the functional outcome(s) of Wnt-induced signals share similarities with cellular redox-mediated networks from the standpoint of MSC biology. Furthermore, there is evidence for a crosstalk between Wnt and redox signalling, which begs the question whether Wnt-mediated differentiation signals involve the intermediary role of reactive oxygen species. In this review, we summarize the impact of Wnt signalling on multi-lineage differentiation of MSCs, and attempt to unravel the intricate interplay between Wnt and redox signals.

Wnt antagonist secreted frizzled-related protein 4 upregulates adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells.

With more than 1.4 billion overweight or obese adults worldwide, obesity and progression of the metabolic syndrome are major health and economic challenges. To address mechanisms of obesity, adipose tissue-derived mesenchymal stem cells (ADSCs) are being studied to detail the molecular mechanisms involved in adipogenic differentiation. Activation of the Wnt signalling pathway has inhibited adipogenesis from precursor cells. In our study, we examined this anti-adipogenic effect in further detail stimulating Wnt with lithium chloride (LiCl) and 6-bromo indirubin 3'oxime (BIO). We also examined the effect of Wnt inhibition using secreted frizzled-related protein 4 (sFRP4), which we have previously shown to be pro-apoptotic, anti-angiogenic, and anti-tumorigenic. Wnt stimulation in LiCl and BIO-treated ADSCs resulted in a significant reduction (2.7-fold and 12-fold respectively) in lipid accumulation as measured by Oil red O staining while Wnt inhibition with sFRP4 induced a 1.5-fold increase in lipid accumulation. Furthermore, there was significant 1.2-fold increase in peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα), and 1.3-fold increase in acetyl CoA carboxylase protein levels. In contrast, the expression of adipogenic proteins (PPARγ, C/EBPα, and acetyl CoA carboxylase) were decreased significantly with LiCl (by 1.6, 2.6, and 1.9-fold respectively) and BIO (by 7, 17, and 5.6-fold respectively) treatments. These investigations demonstrate interplay between Wnt antagonism and Wnt activation during adipogenesis and indicate pathways for therapeutic intervention to control this process.