Proteome-wide analysis of neural stem cell differentiation to facilitate transition to cell replacement therapies.

Neurodegenerative diseases are devastating disorders and the demands on their treatment are set to rise in connection with higher disease incidence. Knowledge of the spatiotemporal profile of cellular protein expression during neural differentiation and definition of a set of markers highly specific for targeted neural populations is a key challenge. Intracellular proteins may be utilized as a readout for follow-up transplantation and cell surface proteins may facilitate isolation of the cell subpopulations, while secreted proteins could help unravel intercellular communication and immunomodulation. This review summarizes the potential of proteomics in revealing molecular mechanisms underlying neural differentiation of stem cells and presents novel candidate proteins of neural subpopulations, where understanding of their functionality may accelerate transition to cell replacement therapies.

Revelation of fibroblast protein commonalities and differences and their possible roles in wound healing and tumourigenesis using co-culture models of cells.

BACKGROUND INFORMATION: The in vitro co-culture models of communication between normal fibroblasts and epithelial cells, such as keratinocytes or squamous cell carcinoma cells of FaDu line representing wound healing or cancer development, were established by non-direct contact between the cells and utilised in this study to examine epithelia-induced changes in overall fibroblast proteome patterns. RESULTS: We were able to select the proteins co-regulated in both models in order to evaluate possible molecular commonalities between wound healing and tumour development. Amongst the most pronounced were the proteins implemented in contractile activity and formation of actin cytoskeleton such as caldesmon, calponin-2, myosin regulatory light-chain 12A and cofilin-1, which were expressed independently of the presence of α-smooth muscle actin. Additionally, proteins altered differently highlighted functional and cellular phenotypes during transition of fibroblasts towards myofibroblasts or cancer-associated fibroblasts. Results showed coordinated regulation of cytoskeleton proteins selective for wound healing which were lost in tumourigenesis model. Vimentin bridged this group of proteins with other regulated proteins in human fibroblasts involved in protein or RNA processing and metabolic regulation. CONCLUSIONS: The findings provide strong support for crucial role of stromal microenvironment in wound healing and tumourigenesis. In particular, epithelia-induced protein changes in fibroblasts offer new potential targets which may lead to novel tailored cancer therapeutic strategies.

Upregulation of IL-6, IL-8 and CXCL-1 production in dermal fibroblasts by normal/malignant epithelial cells in vitro: Immunohistochemical and transcriptomic analyses.

BACKGROUND INFORMATION: Considering an analogy between wound healing and tumour progression, we studied chemokine and cytokine transcription and expression in normal fibroblasts by co-culture and in situ. RESULTS: Whole-genome transcriptome profiling revealed strong upregulation for the interleukin (IL)-6, IL-8 and the chemokine CXCL-1 in in vitro co-cultures of normal fibroblasts with either normal or malignant epithelial cells compared to fibroblast cultures. The same ILs/chemokines were distinctly upregulated in clinical samples of squamous cell carcinoma when compared with paired normal mucosae. Analysis of culture supernatants showed that during the course of co-culture of the fibroblasts with the epithelial cells, IL-6, IL-8 and CXCL-1 were secreted to the culture medium. Experiments with addition of any of the proteins to the culture medium supported the notion that these ILs/chemokines strongly contributed to maintenance of a low-differentiation phenotype of epithelial cells, evaluated by the detection of keratin-8. Simultaneous addition of all factors increased the extent of the effect. These studies were extended by experiments with epithelial cells, either cultured in medium conditioned by preceding use for malignant keratinocytes without and in the presence of normal or cancer-associated fibroblasts or medium containing antibodies against IL-6, IL-8 and CXCL-1. CONCLUSIONS: Our results indicate an analogy between wound healing and tumour growth, support the importance of epithelial-mesenchymal interaction in this model system and establish a potential bio-inspired anticancer therapy.

Development of ovarian hyperstimulation syndrome: interrogation of key proteins and biological processes in human follicular fluid of women undergoing in vitro fertilization.

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication and potentially life-threatening condition resulting from excessive ovarian stimulation during assisted reproductive technologies. Our aim was to identify candidate proteins in follicular fluid (FF) using various proteomic approaches which may help to identify patients at risk of OHSS. We analysed the proteome alterations in FF from patients suffering from severe forms of OHSS (OHSS+) compared with a control group of women without or with only mild signs of OHSS (OHSS-). The 12 abundant proteins of FF were removed using an immunoaffinity system. Pools of remaining depleted proteins were applied to the two-dimensional (2D) electrophoresis and 2D liquid chromatography and proteins in differentially expressed protein spots/fractions were identified by mass spectrometry. Among a total of 19 candidate proteins differentially expressed (P< 0.05) between OHSS+ and OHSS- FF samples, three proteins, namely ceruloplasmin, complement C3 and kininogen-1, were found using both 2D techniques. Computer modelling highlighted the important role of kininogen-1 as an anchor for mediated interactions with other identified proteins including ferritin light chain and ceruloplasmin, hepatocyte growth factor-like protein, as well as complement C3 and gelsolin, thus linking various biological processes including inflammation and angiogenesis, iron transport and storage, blood coagulation, innate immunity, cell adhesion and actin filament polymerization. The delineation of such processes may allow the development of informed corrective therapeutic intervention in patients at risk of OHSS and a set of key proteins of the FF may be helpful as potential biomarkers for monitoring IVF therapy.

Proteome mining of human follicular fluid reveals a crucial role of complement cascade and key biological pathways in women undergoing in vitro fertilization.

In vitro fertilization (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionally, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrome. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multidimensional protein fractionation allowed the study of middle- and lower-abundance proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process.

Revelation of the IFNα, IL-10, IL-8 and IL-1ß as promising biomarkers reflecting immuno-pathological mechanisms in porcine Huntington's disease model.

Studies on Huntington's disease (HD) demonstrated altered immune response in HD gene carriers. Using multiplexing immunoassay, we simultaneously investigated seven cytokines in secretomes of microglia and blood monocytes, cerebrospinal fluid (CSF) and serum collected from transgenic HD minipigs at pre-symptomatic disease stage. Decline in IFNα and IL-10 was observed in CSF and secretome of microglia whilst elevated IL-8 and IL-1ß levels were secreted by microglia. Additionally, IL-8 was increased in serum. The proportion of mutant huntingtin in microglia may have causative impact on cytokine production. IFNα, IL-10, IL-8 and IL-1ß represent promising biomarkers reflecting immuno-pathological mechanisms in porcine HD model.

Multiplex immunoassays for quantification of cytokines, growth factors, and other proteins in stem cell communication.

Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners).

Challenges of Huntington's disease and quest for therapeutic biomarkers.

Huntington's disease (HD) is the most common inherited neurodegenerative disorder among polyglutamine (polyQ) diseases caused by cytosine-adenine-guanine repeat expansion in exon 1 of the huntingtin gene whose translation results in polyQ stretch in the N-terminus of the huntingtin protein (HD protein). This mutation significantly affects huntingtin conformation, proteolysis, PTMs, as well as its ability to bind interacting proteins. As a consequence, a variety of cellular mechanisms such as transcription, mitochondrial energy metabolism, axonal transport, neuronal vulnerability to oxidative stress, neurotransmission, and immune response are altered and involved in the pathogenesis of HD. Promising candidate molecular biomarkers of HD have emerged from proteomic studies. Recent analyses focused on HD protein itself, its PTM, and interacting proteins, which are of great importance for disease course. Furthermore, brain, body fluids, and immune system are intensively studied in order to search for additional proteins with a view to their use as a biomarker(s) or set of biomarkers in clinical trials in HD translational research.