Investigating the role of CRIPTO-1 (TDGF-1) in glioblastoma multiforme U87 cell line.

Cripto-1 has been implicated in a number of human cancers. Although there is high potential for a role of Cripto-1 in glioblastoma multiforme (GBM) pathogenesis and progression, few studies have tried to define its role in GBM. These studies were limited in that Cripto-1 expression was not studied in detail in relation to markers of cancer initiation and progression. Therefore, these correlative studies allowed limited interpretation of Criptos-1's effect on the various aspects of GBM development using the U87 GBM cell line. In this study, we sought to delineate the role of Cripto-1 in facilitating pathogenesis, stemness, proliferation, invasion, migration and angiogenesis in GBM. Our findings show that upon overexpressing Cripto-1 in U87 GBM cells, the stemness markers Nanog, Oct4, Sox2, and CD44 increased expression. Similarly, an increase in Ki67 was observed demonstrating Cripto-1's potential to induce cellular proliferation. Likewise, we report a novel finding that increased expression of the markers of migration and invasion, Vimentin and Twist, correlated with upregulation of Cripto-1. Moreover, Cripto-1 exposure led to VEGFR-2 overexpression along with higher tube formation under conditions promoting endothelial growth. Taken together our results support a role for Cripto-1 in the initiation, development, progression, and maintenance of GBM pathogenesis. The data presented here are also consistent with a role for Cripto-1 in the re-growth and invasive growth in GBM. This highlights its potential use as a predictive and diagnostic marker in GBM as well as a therapeutic target.

Assessing stemness and proliferation properties of the newly established colon cancer 'stem' cell line, CSC480 and novel approaches to identify dormant cancer cells.

To date two questions that remain unanswered regarding cancer are the following: i) how is it initiated, and ii) what is the role that cancer stem cells (CSCs) play in the disease process? Understanding the biology of CSCs and how they are generated is pivotal for the development of successful treatment regimens. To date, the lack of a representative cell model has prevented the successful identification and eradication of CSCs in vivo. The current methods of CSC identification are dependent on the protocol used to generate these cells, which has introduced variation and made the identification process more complicated. Furthermore, the list of possible markers is increasing in complexity. This is further confounded by the fact that there is insufficient information to determine whether the cells these markers detect are truly self­renewing stem cells or, instead, progenitor cells. In the present study, we investigated a novel cell line model, CSC480, which can be employed to assess CSC markers and for testing novel therapeutic regimens. CSC480 cells have been revealed to express markers of CSCs such as CD44, ALDH1 and Sox2, that have lower expression in the SW480 cell line. CSC480 cells also expressed higher levels of the cancer resistance marker, ABCG2 and had higher proliferative and growth capacity than SW480 cells. In the present study, we also evaluated a novel approach to identify different cell types present in heterogeneous cancer cell populations according to their proliferative ability using the proliferation marker 5­ethynyl­2'­deoxyuridine (EdU). Furthermore, using EdU, we identified dormant cells with a modified label­retaining cell (LRC) protocol. Through this novel LRC method, we assessed newly discovered markers of stemness to ascertain their capability to identify quiescent from dividing CSCs. In conclusion, the CSC480 cell line was an important model to be used in unravelling the underlying mechanisms that control fast­dividing and partially self­renewing stem cells (SCs) that may give rise to cancer.

Structural characterisation of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma (BL) Daudi cells by NMR spectroscopy.

Siglec-2 undergoes constitutive endocytosis and is a drug target for autoimmune diseases and B cell-derived malignancies, including hairy cell leukaemia, marginal zone lymphoma, chronic lymphocytic leukaemia and non-Hodgkin's lymphoma (NHL). An alternative to current antibody-based therapies is the use of liposomal nanoparticles loaded with cytotoxic drugs and decorated with Siglec-2 ligands. We have recently designed the first Siglec-2 ligands (9-biphenylcarboxamido-4-meta-nitrophenyl-carboxamido-Neu5Acα2Me, 9-BPC-4-mNPC-Neu5Acα2Me) with simultaneous modifications at C-4 and C-9 position. In the current study we have used Saturation Transfer Difference (STD) NMR spectroscopy to monitor the binding of 9-BPC-4-mNPC-Neu5Acα2Me to Siglec-2 present on intact Burkitt's lymphoma Daudi cells. Pre-treatment of cells with periodate resulted in significantly higher STD NMR signal intensities for 9-BPC-4-mNPC-Neu5Acα2Me as the cells were more susceptible to ligand binding because cis-binding on the cell surface was removed. Quantification of STD NMR effects led to a cell-derived binding epitope of 9-BPC-4-mNPC-Neu5Acα2Me that facilitated the design and synthesis of C-2, C-3, C-4 and C-9 tetra-substituted Siglec-2 ligands showing an 88-fold higher affinity compared to 9-BPC-Neu5Acα2Me. This is the first time a NMR-based binding study of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma Daudi cells has been described that might open new avenues in developing tailored therapeutics and personalised medicine.

A patient-derived olfactory stem cell disease model for ataxia-telangiectasia.

The autosomal recessive disorder ataxia-telangiectasia (A-T) is characterized by genome instability, cancer predisposition and neurodegeneration. Although the role of ataxia-telangiectasia mutated (ATM) protein, the protein defective in this syndrome, is well described in the response to DNA damage, its role in protecting the nervous system is less clear. We describe the establishment and characterization of patient-specific stem cells that have the potential to address this shortcoming. Olfactory neurosphere (ONS)-derived cells were generated from A-T patients, which expressed stem cell markers and exhibited A-T molecular and cellular characteristics that included hypersensitivity to radiation, defective radiation-induced signaling and cell cycle checkpoint defects. Introduction of full-length ATM cDNA into these cells corrected defects in the A-T cellular phenotype. Gene expression profiling and pathway analysis revealed defects in multiple cell signaling pathways associated with ATM function, with cell cycle, cell death and DNA damage response pathways being the most significantly dysregulated. A-T ONS cells were also capable of differentiating into neural progenitors, but they were defective in neurite formation, number of neurites and length of these neurites. Thus, ONS cells are a patient-derived neural stem cell model that recapitulate the phenotype of A-T, do not require genetic reprogramming, have the capacity to differentiate into neurons and have potential to delineate the neurological defect in these patients.

A patient-derived stem cell model of hereditary spastic paraplegia with SPAST mutations.

Hereditary spastic paraplegia (HSP) leads to progressive gait disturbances with lower limb muscle weakness and spasticity. Mutations in SPAST are a major cause of adult-onset, autosomal-dominant HSP. Spastin, the protein encoded by SPAST, is a microtubule-severing protein that is enriched in the distal axon of corticospinal motor neurons, which degenerate in HSP patients. Animal and cell models have identified functions of spastin and mutated spastin but these models lack the gene dosage, mutation variability and genetic background that characterize patients with the disease. In this study, this genetic variability is encompassed by comparing neural progenitor cells derived from biopsies of the olfactory mucosa from healthy controls with similar cells from HSP patients with SPAST mutations, in order to identify cell functions altered in HSP. Patient-derived cells were similar to control-derived cells in proliferation and multiple metabolic functions but had major dysregulation of gene expression, with 57% of all mRNA transcripts affected, including many associated with microtubule dynamics. Compared to control cells, patient-derived cells had 50% spastin, 50% acetylated α-tubulin and 150% stathmin, a microtubule-destabilizing enzyme. Patient-derived cells were smaller than control cells. They had altered intracellular distributions of peroxisomes and mitochondria and they had slower moving peroxisomes. These results suggest that patient-derived cells might compensate for reduced spastin, but their increased stathmin expression reduced stabilized microtubules and altered organelle trafficking. Sub-nanomolar concentrations of the microtubule-binding drugs, paclitaxel and vinblastine, increased acetylated α-tubulin levels in patient cells to control levels, indicating the utility of this cell model for screening other candidate compounds for drug therapies.

Variability in the generation of induced pluripotent stem cells: importance for disease modeling.

In the field of disease modeling, induced pluripotent stem cells (iPSCs) have become an appealing choice, especially for diseases that do not have an animal model. They can be generated from patients with known clinical features and compared with cells from healthy controls to identify the biological bases of disease. This study was undertaken to determine the variability in iPSC lines derived from different individuals, with the aim of determining criteria for selecting iPSC lines for disease models. We generated and characterized 18 iPSC lines from eight donors and considered variability at three levels: (a) variability in the criteria that define iPSC lines as pluripotent cells, (b) variability in cell lines from different donors, and (c) variability in cell lines from the same donor. We found that variability in transgene expression and pluripotency marker levels did not prevent iPSCs from fulfilling all other criteria for pluripotency, including teratoma formation. We found low interindividual and interclonal variability in iPSCs that fulfilled the most stringent criteria for pluripotency, with very high correlation in their gene expression profiles. Interestingly, some cell lines exhibited reprogramming instability, spontaneously regressing from a fully to a partially reprogrammed state. This was associated with a low percentage of cells expressing the pluripotency marker stage-specific embryonic antigen-4. Our study shows that it is possible to define a similar "ground state" for each cell line as the basis for making patient versus control comparisons, an essential step in order to identify disease-associated variability above individual and cell line variability.

Multipotent stem cells from adult olfactory mucosa.

Multipotent stem cells are thought to be responsible for the generation of new neurons in the adult brain. Neurogenesis also occurs in an accessible part of the nervous system, the olfactory mucosa. We show here that cells from human olfactory mucosa generate neurospheres that are multipotent in vitro and when transplanted into the chicken embryo. Cloned neurosphere cells show this multipotency. Multipotency was evident without prior culture in vitro: cells dissociated from adult rat olfactory mucosa generate leukocytes when transplanted into bone marrow-irradiated hosts, and cells dissociated from adult mouse olfactory epithelium generated numerous cell types when transplanted into the chicken embryo. It is unlikely that these results can be attributed to hematopoietic precursor contamination or cell fusion. These results demonstrate the existence of a multipotent stem-like cell in the olfactory mucosa useful for autologous transplantation therapies and for cellular studies of disease.