The Effect of Wnt Pathway Modulators on Human iPSC-Derived Pancreatic Beta Cell Maturation

Current published protocols for targeted differentiation of human stem cells toward pancreatic β-cells fail to deliver sufficiently mature cells with functional properties comparable to human islet β-cells. We aimed to assess whether Wnt-modulation could promote the final protocol stages of β-cell maturation, building our hypothesis on our previous findings of Wnt activation in immature hiPSC-derived stage 7 (S7) cells compared to adult human islets and with recent data reporting a link between Wnt/PCP and in vitro β-cell maturation. In this study, we stimulated canonical and non-canonical Wnt signaling in hiPSC-derived S7 cells using syntetic proteins including WNT3A, WNT4, WNT5A and WNT5B, and we inhibited endogenous Wnt signaling with the Tankyrase inhibitor G007-LK (TKi). Whereas neither canonical nor non-canonical Wnt stimulation alone was able to mature hiPSC-derived S7 cells, WNT-inhibition with TKi increased the fraction of monohormonal cells and global proteomics of TKi-treated S7 cells showed a proteomic signature more similar to adult human islets, suggesting that inhibition of endogenous Wnt contributes toward final β-cell maturation

In-depth characterization of the cerebrospinal fluid (CSF) proteome displayed through the CSF proteome resource (CSF-PR)

In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics.publishedVersio

Arachnoid cysts do not contain cerebrospinal fluid: A comparative chemical analysis of arachnoid cyst fluid and cerebrospinal fluid in adults

<p>Abstract</p> <p>Background</p> <p>Arachnoid cyst (AC) fluid has not previously been compared with cerebrospinal fluid (CSF) from the same patient. ACs are commonly referred to as containing "CSF-like fluid". The objective of this study was to characterize AC fluid by clinical chemistry and to compare AC fluid to CSF drawn from the same patient. Such comparative analysis can shed further light on the mechanisms for filling and sustaining of ACs.</p> <p>Methods</p> <p>Cyst fluid from 15 adult patients with unilateral temporal AC (9 female, 6 male, age 22-77y) was compared with CSF from the same patients by clinical chemical analysis.</p> <p>Results</p> <p>AC fluid and CSF had the same osmolarity. There were no significant differences in the concentrations of sodium, potassium, chloride, calcium, magnesium or glucose. We found significant elevated concentration of phosphate in AC fluid (0.39 versus 0.35 mmol/L in CSF; <it>p </it>= 0.02), and significantly reduced concentrations of total protein (0.30 versus 0.41 g/L; <it>p </it>= 0.004), of ferritin (7.8 versus 25.5 ug/L; <it>p </it>= 0.001) and of lactate dehydrogenase (17.9 versus 35.6 U/L; <it>p </it>= 0.002) in AC fluid relative to CSF.</p> <p>Conclusions</p> <p>AC fluid is not identical to CSF. The differential composition of AC fluid relative to CSF supports secretion or active transport as the mechanism underlying cyst filling. Oncotic pressure gradients or slit-valves as mechanisms for generating fluid in temporal ACs are not supported by these results.</p

Characterization of arachnoid cysts using clinical chemistry, qualitative and quantitative proteomics

Arachnoid cyst are benign intracranial lesions with a reported prevalence up to 1.1 % in the population. The origin of such cysts and the mechanisms of filling and sustaining are poorly understood. The aim of the thesis was to characterize the arachnoid cyst fluid and compare it with cerebrospinal fluid from the same individuals to evaluate the content of arachnoid cysts, as well as to gain further knowledge of the mechanisms of filling and sustaining of such cysts. Patients were recruited prior to elective surgery for fenestration of symptomatic arachnoid cysts in the temporal fossa and arachnoid cyst fluid and cerebrospinal fluid was collected with written informed consent from 19 patients. In Paper I the content of arachnoid cyst fluid and cerebrospinal fluid from the same patients were compared by clinical chemistry. The protein content of arachnoid cyst fluid is reduced relative to cerebrospinal fluid, while the concentration of phosphate is elevated. The results from this evaluation indicated that arachnoid cyst fluid is not identical to cerebrospinal fluid. In Paper II the protein content in arachnoid cyst fluid from 15 patients was evaluated by qualitative proteomics and the findings were compared with published databases of plasma and cerebrospinal fluid. These comparisons indicated that the arachnoid cyst fluid proteome was similar to cerebrospinal fluid, but dissimilar to plasma. In Paper III we performed a quantitative comparison of the proteomes of arachnoid cyst fluid and cerebrospinal fluid for five patients. 348 proteins were quantified in individual patients, and 1425 proteins in a pool of the same patients using an iTRAQ-strategy combined with extensive fractionation. We identify differences between the fluids, but currently we are not able to elute the biological significance. Searched against DNA and mRNA-data, we find some differences, but not in patterns of biological significance. This is the first quantitative proteomics comparison of AC fluid and CSF. In conclusion, the work presented in this thesis indicates that AC fluid is similar, but not identical, to CSF. Results do not support oncotic filling or valves as mechanisms for filling and sustaining of arachnoid cysts but rather an active or selective mechanism for filling

Quantitative proteomics comparison of arachnoid cyst fluid and cerebrospinal fluid collected perioperatively from arachnoid cyst patients

Background: There is little knowledge concerning the content and the mechanisms of filling of arachnoid cysts. The aim of this study was to compare the protein content of arachnoid cysts and cerebrospinal fluid by quantitative proteomics to increase the understanding of arachnoid cysts. Methods: Arachnoid cyst fluid and cerebrospinal fluid from five patients were analyzed by quantitative proteomics in two separate experiments. In a label-free experiment arachnoid cyst fluid and cerebrospinal fluid samples from individual patients were trypsin digested and analyzed by Orbitrap mass spectrometry in a label-free manner followed by data analysis using the Progenesis software. In the second proteomics experiment, a patient sample pooling strategy was followed by MARS-14 immunodepletion of high abundant proteins, trypsin digestion, iTRAQ labelling, and peptide separation by mix-phase chromatography followed by Orbitrap mass spectrometry analysis. The results from these analyzes were compared to previously published mRNA microarray data obtained from arachnoid membranes. Results: We quantified 348 proteins by the label-free individual patient approach and 1425 proteins in the iTRAQ experiment using a pool from five patients of arachnoid cyst fluid and cerebrospinal fluid. This is by far the largest number of arachnoid cyst fluid proteins ever identified, and the first large-scale quantitative comparison between the protein content of arachnoid cyst fluid and cerebrospinal fluid from the same patients at the same time. Consistently in both experiment, we found 22 proteins with significantly increased abundance in arachnoid cysts compared to cerebrospinal fluid and 24 proteins with significantly decreased abundance. We did not observe any molecular weight gradient over the arachnoid cyst membrane. Of the 46 proteins we identified as differentially abundant in our study, 45 were also detected from the mRNA expression level study. None of them were previously reported as differentially expressed. We did not quantify any of the proteins corresponding to gene products from the ten genes previously reported as differentially abundant between arachnoid cysts and control arachnoid membranes. Conclusions: From our experiments, the protein content of arachnoid cyst fluid and cerebrospinal fluid appears to be similar. There were, however, proteins that were significantly differentially abundant between arachnoid cyst fluid and cerebrospinal fluid. This could reflect the possibility that these proteins are affected by the filling mechanism of arachnoid cysts or are shed from the membranes into arachnoid cyst fluid. Our results do not support the proposed filling mechanisms of oncotic pressure or valves

Mapping Proteome Changes in Microsatellite Stable, Recurrent Colon Cancer Reveals a Significant Immune System Signature

Background/Aim: Better stratification of the risk of relapse will help select the right patients for adjuvant treatment and improve targeted therapies for patients with colon cancer. Materials and Methods: To understand why a subset of tumors relapse, we compared the proteome of two groups of patients with colon cancer with similar stage, stratified based on the presence or absence of recurrence. Results: Using tumor biopsies from the primary operation, we identified dissimilarity between recurrent and nonrecurrent mismatch satellite stable colon cancer and found that signaling related to immune activation and inflammation was associated with relapse. Conclusion: Immune modulation may have an effect on mismatch satellite stable colon cancer. At present, immune therapy is offered primarily to microsatellite instable colon cancer. Hopefully, immune therapy in mismatch satellite stable colon cancer beyond PD-1 and PD-L1 inhibitors can be implemented.publishedVersio

The effect of WnT pathway modulators on human iPSC-derived pancreatic beta cell maturation

Current published protocols for targeted differentiation of human stem cells toward pancreatic β-cells fail to deliver sufficiently mature cells with functional properties comparable to human islet β-cells. We aimed to assess whether Wnt-modulation could promote the final protocol stages of β-cell maturation, building our hypothesis on our previous findings of Wnt activation in immature hiPSC-derived stage 7 (S7) cells compared to adult human islets and with recent data reporting a link between Wnt/PCP and in vitro β-cell maturation. In this study, we stimulated canonical and non-canonical Wnt signaling in hiPSC-derived S7 cells using syntetic proteins including WNT3A, WNT4, WNT5A and WNT5B, and we inhibited endogenous Wnt signaling with the Tankyrase inhibitor G007-LK (TKi). Whereas neither canonical nor non-canonical Wnt stimulation alone was able to mature hiPSC-derived S7 cells, WNT-inhibition with TKi increased the fraction of monohormonal cells and global proteomics of TKi-treated S7 cells showed a proteomic signature more similar to adult human islets, suggesting that inhibition of endogenous Wnt contributes toward final β-cell maturation

Novel protein signatures suggest progression to muscular invasiveness in bladder cancer.

Patients with bladder cancer need frequent controls over long follow-up time due to high recurrence rate and risk of conversion to muscle invasive cancer with poor prognosis. We identified cancer-related molecular signatures in apparently healthy bladder in patients with subsequent muscular invasiveness during follow-up. Global proteomics of the normal tissue biopsies revealed specific proteome fingerprints in these patients prior to subsequent muscular invasiveness. In these presumed normal samples, we detected modulations of proteins previously associated with different cancer types. This study indicates that analyzing apparently healthy tissue of a cancer-invaded organ may suggest disease progression

In-depth characterization of the cerebrospinal fluid (CSF) proteome displayed through the CSF proteome resource (CSF-PR)

In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics