MicroRNA expression signatures and their correlation with clinicopathological features in glioblastoma multiforme.

The increasing interest in identifying molecular biomarkers to determine patient prognosis in glioblastoma multiforme (GBM) has resulted in several microRNA (miRNA)-based signatures able to predict progression-free and overall survival. However, the coherency between these signatures is small, and correlations to clinicopathological features other than survival are seldom seen. The aim of this study was to identify any significant relationship between miRNA signatures and clinicopathological data by combining pathological features with miRNA and mRNA analysis in fourteen GBM patients. In total, 161 miRNAs were shown to cluster the GBM tumor samples into long- and short-term-surviving patients. Many of these miRNAs were associated with differential expression in GBM, including a number of miRNAs shown to confer risk or protection with respect to clinical outcome and to modulate the mesenchymal mode of migration and invasion. An inverse relationship between miR-125b and nestin expression was identified and correlated with overall survival in GBM patients, eloquently illustrating how clinicopathological findings and molecular profiling may be a relevant combination to predict patient outcome. The intriguing finding that many of the differentially expressed miRNAs contained exosome-packaging motifs in their mature sequences suggests that we must expand our view to encompass the complex intercellular communication in order to identify molecular prognostic biomarkers and to increase our knowledge in the field of GBM pathogenesis.

A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy.

Exosomes denote a class of secreted nanoparticles defined by size, surface protein and lipid composition, and the ability to carry RNA and proteins. They are important mediators of intercellular communication and regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest them to be important both for diagnostic and therapeutic purposes, prompting the idea of using exosomes as drug delivery vehicles, especially for gene therapy. This review covers the current status of evidence presented in the field of exosome-based drug delivery systems. Components for successful exosome-based drug delivery, such as choice of donor cell, therapeutic cargo, use of targeting peptide, loading method and administration route are highlighted and discussed with a general focus pertaining to the results obtained in models of different cancer types. In addition, completed and on-going clinical trials are described, evaluating exosome-based therapies for the treatment of different cancer types. Due to their endogenous origin, exosome-based drug delivery systems may have advantages in the treatment of cancer, but their design needs further refinement to justify their usage on the clinical scale.

MicroRNA expression signatures determine prognosis and survival in glioblastoma multiforme--a systematic overview.

Despite advances in our knowledge about glioblastoma multiforme (GBM) pathology, clinical challenges still lie ahead with respect to treatment in GBM due to high prevalence, poor prognosis, and frequent tumor relapse. The implication of microRNAs (miRNAs) in GBM is a rapidly expanding field of research with the aim to develop more targeted molecular therapies. This review aims to present a comprehensive overview of all the available literature, evaluating miRNA signatures as a function of prognosis and survival in GBM. The results are presented with a focus on studies derived from clinical data in databases and independent tissue cohorts where smaller samples sizes were investigated. Here, miRNA associated to longer survival (protective) and miRNA with shorter survival (risk-associated) have been identified and their signatures based on different prognostic attributes are described. Finally, miRNAs associated with disease progression or survival in several studies are identified and functionally described. These miRNAs may be valuable for future determination of patient prognosis and could possibly serve as targets for miRNA-based therapies, which hold a great potential in the treatment of this severe malignant disease.

Cripto-1 expression in glioblastoma multiforme.

Human glioblastoma multiforme (GBM) is an aggressive cancer with a very poor prognosis. Cripto-1 (CR-1) has a key regulatory role in embryogenesis, while in adult tissue re-expression of CR-1 has been correlated to malignant progression in solid cancers of non-neuronal origin. As CR-1 expression has yet to be described in cerebral cancer and CR-1 is regulated by signaling pathways dysregulated in GBM, we aimed to investigate CR-1 in the context of expression in GBM. The study was performed using enzyme-linked immunosorbent assay (ELISA), Western blotting, polymerase chain reaction (PCR) and immunohistochemistry to analyze the blood and tissue from 28 GBM and 4 low-grade glioma patients. Within the patient cohort, we found high CR-1 protein levels in blood plasma to significantly correlate with a shorter overall survival. We identified CR-1 in different areas of GBM tissue, including perivascular tumor cells, and in endothelial cells. Collectively, our data suggest that CR-1 could be a prognostic biomarker for GBM with the potential of being a therapeutic target.

Targeted antiepidermal growth factor receptor (cetuximab) immunoliposomes enhance cellular uptake in vitro and exhibit increased accumulation in an intracranial model of glioblastoma multiforme.

Therapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%. Nanoparticles consisting of liposome-based therapeutics are provided against a variety of cancer types including GBM, but available liposomal formulations are provided without targeting moieties, which increases the dosing demands to reach therapeutic concentrations with risks of side effects. We prepared PEGylated immunoliposomes (ILs) conjugated with anti-human epidermal growth factor receptor (EGFR) antibodies Cetuximab ( α -hEGFR-ILs). The affinity of the α -hEGFR-ILs for the EGF receptor was evaluated in vitro using U87 mg and U251 mg cells and in vivo using an intracranial U87 mg xenograft model. The xenograft model was additionally analyzed with respect to permeability to endogenous albumin, tumor size, and vascularization. The in vitro studies revealed significantly higher binding of α -hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin. The uptake and internalization of the α -hEGFR-ILs by U87 mg cells were further confirmed by 3D deconvolution analyses. In vivo, the α -hEGFR-ILs accumulated to a higher extent inside the tumor when compared to nonimmune liposomes. The data show that α -hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery.

Defined xenogeneic-free and hypoxic environment provides superior conditions for long-term expansion of human adipose-derived stem cells.

Development and implementation of therapeutic protocols based on stem cells or tissue-engineered products relies on methods that enable the production of substantial numbers of cells while complying with stringent quality and safety demands. In the current study, we aimed to assess the benefits of maintaining cultures of adipose-derived stem cells (ASCs) in a defined culture system devoid of xenogeneic components (xeno-free) and hypoxia over a 49-day growth period. Our data provide evidence that conditions involving StemPro mesenchymal stem cells serum-free medium (SFM) Xeno-Free and hypoxia (5% oxygen concentration) in the culture atmosphere provide a superior proliferation rate compared to a standard growth environment comprised of alpha-modified Eagle medium (A-MEM) supplemented with fetal calf serum (FCS) and ambient air (20% oxygen concentration) or that of A-MEM supplemented with FCS and hypoxia. Furthermore, a flow cytometric analysis and in vitro differentiation assays confirmed the immunophenotype stability and maintained multipotency of ASCs when expanded under xeno-free conditions and hypoxia. In conclusion, our data demonstrate that growth conditions utilizing a xeno-free and hypoxic environment not only provide an improved environment for the expansion of ASCs, but also set the stage as a culture system with the potential broad spectrum utility for regenerative medicine and tissue engineering applications.

[The haematological biobank--an infrastructure for quality assurance, development and research]. / Den hæmatologiske biobank er en infrastruktur til kvalitetssikring, udvikling og forskning.

Archived patient samples of biological material coupled to clinical databases are valuable and hold the potential to improve patient care, quality control, research and development. Biobanks form a catalytic infrastructure which ensures the registration, handling and storage of collected biological material. Here, we describe the Haematology Biobank and stress the significance of a registration database. Together, these form the basis for retrospective validation of clinical progress and prognostic evaluation of new diagnostic methods, which can therefore be more quickly implemented in the clinic.

Adipose-derived stem cells from the brown bear (Ursus arctos) spontaneously undergo chondrogenic and osteogenic differentiation in vitro.

In the den, hibernating brown bears do not develop tissue atrophy or organ damage, despite almost no physical activity. Mesenchymal stem cells could play an important role in tissue repair and regeneration in brown bears. Our objective was to determine if adipose tissue-derived stem cells (ASCs) can be recovered from wild Scandinavian brown bears and characterize their differentiation potential. Following immobilization of wild brown bears 7-10 days after leaving the den in mid-April, adipose tissue biopsies were obtained. ASCs were recovered from 6 bears, and shown to be able to undergo adipogenesis and osteogenesis in monolayer cultures and chondrogenesis in pellet cultures. Remarkably, when grown in standard cell culture medium in monolayer cultures, ASCs from yearlings spontaneously formed bone-like nodules surrounded by cartilaginous deposits, suggesting differentiation into osteogenic and chondrogenic lineages. This ability appears to be lost gradually with age. This is the first study to demonstrate stem cell recovery and growth from brown bears, and it is the first report of ASCs spontaneously forming extracellular matrix characteristic of bone and cartilage in the absence of specific inducers. These findings could have implications for the use of hibernating brown bears as a model to study disuse osteoporosis.

Isolation and expansion of adipose-derived stem cells for tissue engineering.

For treatment of cardiac failure with bone marrow-derived mesenchymal stem cells, several clinical trials are ongoing. However, more attention is gathering on the use of adipose tissue-derived stem cells (ASCs). This paper describes the optimization of isolation and propagation of ASCs for subsequent clinical use. In the isolation step, several enzymes were compared with respect to yield of nucleated cells and precursor cells. Our results showed, that the interdonor variablility was greater than differences between individual enzymes. For propagation of cells, different types of media, sera and serum replacers were evaluated regarding their ability to support cell growth and preserve differentiation potential. Most of serum replacers proved inferior to fetal calf serum. Among the media tested, modified Eagle's media alpha was superior in promoting cell growth while preserving the ability to differentiate. Also, the effect of cell seeding density and hypoxic culture was evaluated. In this study, we show that it is possible to maximize cell yield regardless of donor individual characteristics by simple manipulations of media composition, cell seeding density and gaseous environment.

Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells.

BACKGROUND AIMS: Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media. METHODS: The effects of prolonged hypoxic culture on growth and pro-angiogenic properties were investigated using human ASC cultured at 1%, 5% and 21% oxygen. The effect of trypsinization on the expression of pro-angiogenic genes was also determined. RESULTS: Trypsinization induced up-regulation of the vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) genes independent of oxygen concentration. The expression of VEGF and IGF-1 was up-regulated in ASC cultured at 1% oxygen for 13 days compared with 4 days. The VEGF concentration in ASC-conditioned media was higher after prolonged hypoxic culture compared with short-term culture, while the IGF-1 and chemokine (CXC motif) ligand 12 (CXCL12) concentrations were unchanged. The VEGF receptor blocker SU5416 abolished angiogenesis in a cultured rat aortic ring model. Media from cells exposed to hypoxia increased angiogenesis, an effect that was dependent on factors other than just the VEGF concentration in the added media. CONCLUSIONS: Optimization of the angiogenic potential of stem cell-based therapy in the treatment of vascular disease is important. We have demonstrated that prolonged hypoxic culture and trypsinization augment the therapeutic angiogenic potential of ASC.

[Use of translational database at clinical department]. / Brug af en translationel database i en klinisk afdeling.

In haematology it is assumed that integrative analysis of global gene expression, protein and cell profiles as well as clinical data will lead to the development of new diagnostic, prognostic and predictive methods. A translational database system registering and combining all data and clinical observations about the patient is therefore needed. It is expected that along with automated prediction and prognosis tools, such a database system may have the potential to assist the development of new machine-based diagnostic decision-making processes.

Cancer stem cells and the cellular hierarchy in haematological malignancies.

Malignancies in the haematopoietic system seem to depend on a small subset of so-called cancer stem cells (CSC) for their continued growth and progression - this was first described as the "sleeper-feeder theory" for leukaemia. The leukaemia stem cell was the first of such subsets to be described although the origins of these cells have been difficult to dissect. Consequently, their biology is not fully elucidated, which also holds true for the normal-tissue counterparts. The stem cell concept describes stem cells to be of low frequency, self renewing and with multilineage potential based on phenomenology - a definition which may not hold strictly true for CSCs when studied in animals and humans in vivo and in vitro. Several studies have analysed the cellular hierarchy of the haematopoietic system by cell sorting of few and even single cells, tracking acquired genetic changes and performing transplantation model studies to document subsets within the differentiating hierarchy as potential CSC compartments. In leukaemia the CSC has been described in the bone marrow compartment of haematopoietic stem cells (HSC); however, in other bone marrow disorders like multiple myeloma it is likely that the cell of origin is a more differentiated cell, like post-germinal memory B cells or plasmablasts. Studies performed so far have even indicated that the genetic events may occur in different B cell subsets in accordance with the stepwise oncogenesis of the disease. Although our understanding of the nature and biology of these initiating cells remains unknown, the obvious existence of such cells has implications for understanding initial malignant transformation and disease metastasis or progression and, most important, the selection of individualised therapeutic strategies targeting the subsets harbouring the CSC function. In the present review on stem cells in haematological malignancies we have focused on two topics, first, describing the stem cell concept in health and disease, and its "phenomenology", and second, describing the CSC compartments in leukaemia and multiple myeloma.

Effect of growth media and serum replacements on the proliferation and differentiation of adipose-derived stem cells.

BACKGROUND AIMS: Studies of mesenchymal stromal cells (MSC) from BM and adipose tissue have demonstrated similar differentiation potentials along the adipo-, osteo- and chondrogenic lineages. While most clinical trials have been performed using BM-derived MSC, the focus is shifting toward the use of stem cells derived from fat tissue. The aim of the current investigation was to define optimal culture conditions that would facilitate clinical use of adipose-derived stem cells (ASC). METHODS: Different types and concentrations of serum replacers and basal media were tested with respect to the optimal expansion and subsequent differentiation of primary human ASC. The effect of initial seeding density on the growth of ASC was also determined. RESULTS: While several of the serum replacements proved to be clearly inferior to fetal calf serum (FCS) in promoting ASC growth, the knockout serum replacement (KOSR) had expansion properties similar to those of FCS. However, with respect to the capacity to support adipo-, osteo- and chondrogenic differentiation, KOSR proved to be less consistent than FCS. Among the media formulations, modified Eagle medium alpha supported a significantly faster cell expansion than the other basal media while still maintaining the full differentiation potential of ASC. Regarding the plating density most favorable for rapid expansion, we found that initial plating densities ranging from 100 to 200 cell/cm(2) resulted in significantly shorter doubling times than plating densities both below and above that range. CONCLUSIONS: Identification of the optimal basal medium and serum replacer, together with the most favorable plating density, will facilitate cell-based and tissue-engineering applications employing ASC in pre-clinical and clinical settings.

Instability of standard PCR reference genes in adipose-derived stem cells during propagation, differentiation and hypoxic exposure.

BACKGROUND: For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained. RESULTS: In this study, we have compared the gene expression levels of a panel of twelve widely used reference genes during hypoxic culture, osteogenic and chondrogenic differentiation, and passaging of primary human ASCs. We found that several of the commonly used reference genes including 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were unsuitable for normalization in the conditions we tested, whereas tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ), TATAA-box binding protein (TBP), beta-glucuronidase (GUSB) were the most stable across all conditions. CONCLUSION: When determining gene expression levels in adipose-derived stem cells, we recommend normalizing transcription levels to the geometric mean of YMHAZ, TBP and GUSB.

Comparative analysis of highly defined proteases for the isolation of adipose tissue-derived stem cells.

BACKGROUND: Before the potential of adipose tissue-derived stem cells can fully be exploited for a broad scope of tissue-engineering and cell-based therapeutical applications, an effective and reproducible method for isolation is needed. AIM: To comparatively analyze five highly defined protease formulations, Blendzyme 1-4, liberase H1 and a crude collagenase mixture in the course of digestion that consisted of three 1-h intervals. METHODS: The resulting digests of human adipose tissue aspirates were evaluated for the yield of nucleated cells, viability and frequency of specific lineages, in particular CD90, CD34 and CD45, by flow cytometry. The functionality of the cells was assessed as to the colony-forming capacity in limiting dilution assays. RESULTS: Based on all evaluation criteria, Blendzymes 1 and 2 and liberase H1 demonstrated a superior performance and highest consistency. Blendzyme 3 clearly underperformed compared with all other enzymes, and the performance of the rest of enzymes appeared erratic. As for the length of digestion, a 2-h interval appeared optimal when weighing both the yield and functionality of the cells in the stromal vascular fractions obtained from different adipose tissue samples. CONCLUSION: Our results demonstrate that the highly purified proteases provide a valuable alternative to crude collagenase preparations, especially in scenarios where a high definition and reproducibility of the digestion process is of importance.