Spatial Environment Affects HNF4A Mutation-Specific Proteome Signatures and Cellular Morphology in hiPSC-Derived ß-Like Cells.

Studies of monogenic diabetes are particularly useful because we can gain insight into the molecular events of pancreatic ß-cell failure. Maturity-onset diabetes of the young 1 (MODY1) is a form of monogenic diabetes caused by a mutation in the HNF4A gene. Human-induced pluripotent stem cells (hiPSCs) provide an excellent tool for disease modeling by subsequently directing differentiation toward desired pancreatic islet cells, but cellular phenotypes in terminally differentiated cells are notoriously difficult to detect. Re-creating a spatial (three-dimensional [3D]) environment may facilitate phenotype detection. We studied MODY1 by using hiPSC-derived pancreatic ß-like patient and isogenic control cell lines in two different 3D contexts. Using size-adjusted cell aggregates and alginate capsules, we show that the 3D context is critical to facilitating the detection of mutation-specific phenotypes. In 3D cell aggregates, we identified irregular cell clusters and lower levels of structural proteins by proteome analysis, whereas in 3D alginate capsules, we identified altered levels of glycolytic proteins in the glucose sensing apparatus by proteome analysis. Our study provides novel knowledge on normal and abnormal function of HNF4A, paving the way for translational studies of new drug targets that can be used in precision diabetes medicine in MODY.

Leptin Receptor Signaling Regulates Protein Synthesis Pathways and Neuronal Differentiation in Pluripotent Stem Cells.

The role of leptin receptor (OB-R) signaling in linking pluripotency with growth and development and the consequences of dysfunctional leptin signaling on progression of metabolic disease is poorly understood. Using a global unbiased proteomics approach we report that embryonic fibroblasts (MEFs) carrying the db/db mutation exhibit metabolic abnormalities, while their reprogrammed induced pluripotent stem cells (iPSCs) show altered expression of proteins involved in embryonic development. An upregulation in expression of eukaryotic translation initiation factor 4e (Eif4e) and Stat3 binding to the Eif4e promoter was supported by enhanced protein synthesis in mutant iPSCs. Directed differentiation of db/db iPSCs toward the neuronal lineage showed defects. Gene editing to correct the point mutation in db/db iPSCs using CRISPR-Cas9, restored expression of neuronal markers and protein synthesis while reversing the metabolic defects. These data imply a direct role for OB-R in regulating metabolism in embryonic fibroblasts and key developmental pathways in iPSCs.

Bioinformatic Analyses of miRNA-mRNA Signature during hiPSC Differentiation towards Insulin-Producing Cells upon HNF4α Mutation.

Mutations in the hepatocyte nuclear factor 4α (HNF4α) gene affect prenatal and postnatal pancreas development, being characterized by insulin-producing ß-cell dysfunction. Little is known about the cellular and molecular mechanisms leading to ß-cell failure as result of HNF4α mutation. In this study, we compared the miRNA profile of differentiating human induced pluripotent stem cells (hiPSC) derived from HNF4α+/Δ mutation carriers and their family control along the differentiation timeline. Moreover, we associated this regulation with the corresponding transcriptome profile to isolate transcript-miRNA partners deregulated in the mutated cells. This study uncovered a steep difference in the miRNA regulation pattern occurring during the posterior foregut to pancreatic endoderm transition, defining early and late differentiation regulatory windows. The pathway analysis of the miRNAome-transcriptome interactions revealed a likely gradual involvement of HNF4α+/Δ mutation in p53-mediated cell cycle arrest, with consequences for the proliferation potential, survival and cell fate acquisition of the differentiating cells. The present study is based on bioinformatics approaches and we expect that, pending further experimental validation, certain miRNAs deregulated in the HNF4α+/Δ cells would prove useful for therapy.

Matrix decoded - A pancreatic extracellular matrix with organ specific cues guiding human iPSC differentiation.

The extracellular matrix represents a dynamic microenvironment regulating essential cell functions in vivo. Tissue engineering approaches aim to recreate the native niche in vitro using biological scaffolds generated by organ decellularization. So far, the organ specific origin of such scaffolds was less considered and potential consequences for in vitro cell culture remain largely elusive. Here, we show that organ specific cues of biological scaffolds affect cellular behavior. In detail, we report on the generation of a well-preserved pancreatic bioscaffold and introduce a scoring system allowing standardized inter-study quality assessment. Using multiple analysis tools for in-depth-characterization of the biological scaffold, we reveal unique compositional, physico-structural, and biophysical properties. Finally, we prove the functional relevance of the biological origin by demonstrating a regulatory effect of the matrix on multi-lineage differentiation of human induced pluripotent stem cells emphasizing the significance of matrix specificity for cellular behavior in artificial microenvironments.

In vivo Environment Swiftly Restricts Human Pancreatic Progenitors Toward Mono-Hormonal Identity via a HNF1A/HNF4A Mechanism.

Generating insulin-producing ß-cells from human induced pluripotent stem cells is a promising cell replacement therapy for improving or curing insulin-dependent diabetes. The transplantation of end-stages differentiating cells into living hosts was demonstrated to improve ß-cell maturation. Nevertheless, the cellular and molecular mechanisms outlining the transplanted cells' response to the in vivo environment are still to be properly characterized. Here we use global proteomics and large-scale imaging techniques to demultiplex and filter the cellular processes and molecular signatures modulated by the immediate in vivo effect. We show that in vivo exposure swiftly confines in vitro generated human pancreatic progenitors to single hormone expression. The global proteome landscape of the transplanted cells was closer to native human islets, especially in regard to energy metabolism and redox balance. Moreover, our study indicates a possible link between these processes and certain epigenetic regulators involved in cell identity. Pathway analysis predicted HNF1A and HNF4A as key regulators controlling the in vivo islet-promoting response, with experimental evidence suggesting their involvement in confining islet cell fate following xeno-transplantation.

Dynamic proteome profiling of human pluripotent stem cell-derived pancreatic progenitors.

A comprehensive characterization of the molecular processes controlling cell fate decisions is essential to derive stable progenitors and terminally differentiated cells that are functional from human pluripotent stem cells (hPSCs). Here, we report the use of quantitative proteomics to describe early proteome adaptations during hPSC differentiation toward pancreatic progenitors. We report that the use of unbiased quantitative proteomics allows the simultaneous profiling of numerous proteins at multiple time points, and is a valuable tool to guide the discovery of signaling events and molecular signatures underlying cellular differentiation. We also monitored the activity level of pathways whose roles are pivotal in the early pancreas differentiation, including the Hippo signaling pathway. The quantitative proteomics data set provides insights into the dynamics of the global proteome during the transition of hPSCs from a pluripotent state toward pancreatic differentiation.

Reprogrammed Cells Display Distinct Proteomic Signatures Associated with Colony Morphology Variability.

Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity.

Probing the missing mature ß-cell proteomic landscape in differentiating patient iPSC-derived cells.

MODY1 is a maturity-onset monogenic diabetes, caused by heterozygous mutations of the HNF4A gene. To date the cellular and molecular mechanisms leading to disease onset remain largely unknown. In this study, we demonstrate that insulin-positive cells can be generated in vitro from human induced pluripotent stem cells (hiPSCs) derived from patients carrying a non-sense HNF4A mutation, proving for the first time, that a human HNF4A mutation is neither blocking the expression of the insulin genes nor the development of insulin-producing cells in vitro. However, regardless of the mutation or diabetes status, these insulin-producing cells are immature, a common downfall off most current ß-cell differentiation protocols. To further address the immature state of the cells, in vitro differentiated cells and adult human islets were compared by global proteomic analysis. We report the predicted upstream regulators and signalling pathways characterizing the proteome landscape of each entity. Subsequently, we focused on the molecular components absent or misregulated in the in vitro differentiated cells, to probe the components involved in the deficient in vitro maturation towards fully functional ß-cells. This analysis identified the modulation of key developmental signalling pathways representing potential targets for improving the efficiency of the current differentiation protocols.

Quantitative proteomics suggests decrease in the secretogranin-1 cerebrospinal fluid levels during the disease course of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing-remitting MS (RRMS) patients and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase-3-like protein 1, secretogranin-1 (Sg1), cerebellin-1, neuroserpin, cell surface glycoprotein MUC18, testican-2 and glutamate receptor 4. An independent sample set of 13 early-MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early-MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.

Carboxyl-ester lipase maturity-onset diabetes of the young disease protein biomarkers in secretin-stimulated duodenal juice.

Patients with carboxyl-ester lipase-maturity-onset diabetes of the young (CEL-MODY) display distinct disease stages toward the development of monogenic diabetes and exocrine pancreatic disease. The finding of differentially increased proteins, some related to MAPK signaling, in a discovery proteomics study of secretin-stimulated duodenal juice in three CEL-MODY patients, prompted us to monitor their abundance in an extensive number of CEL-MODY subjects at different disease stages and controls using targeted proteomics. In the current study, we demonstrate the feasibility of selected reaction monitoring assays to quantify protein levels in secretin-stimulated duodenal juice. Furthermore, we define a set of five peptides for potential use as diagnostic tests in CEL-MODY patients. Finally, we propose a further set of seven proteins with a likely pathogenic role in CEL-MODY disease progression.

Effects of blood contamination and the rostro-caudal gradient on the human cerebrospinal fluid proteome.

Over the last years there has been an increased focus on the importance of knowing the effect of pre-analytical influence on the proteomes under study, particularly in the field of biomarker discovery. We present three proteomics studies examining the effect of blood contamination and the rostro-caudal gradient (RCG) on the cerebrospinal fluid (CSF) proteome, in addition to plasma/CSF protein ratios. The studies showed that the central nervous system (CNS) derived proteins appeared to be unaffected by the RCG, while the plasma-derived proteins showed an increase in concentration towards the lumbar area. This implies that the concentration of the plasma-derived proteins in CSF will vary depending on the volume of CSF that is collected. In the CSF samples spiked with blood, 262 of 814 quantified proteins showed an abundance increase of more than 1.5 fold, while 403 proteins had a fold change of less than 1.2 and appeared to be unaffected by blood contamination. Proteins with a high plasma/CSF ratio appeared to give the largest effect on the CSF proteome upon blood contamination. The results give important background information on how factors like blood contamination, RCG and blood-CNS-barrier influences the CSF proteome. This information is particularly important in the field of biomarker discovery, but also for routine clinical measurements. The data from the blood contamination and RCG discovery studies have been deposited to the ProteomeXchange with identifier PXD000401.

Use of stable isotope dimethyl labeling coupled to selected reaction monitoring to enhance throughput by multiplexing relative quantitation of targeted proteins.

In this manuscript, we present a proof-of-concept study for targeted relative protein quantitation workflow using chemical labeling in the form of dimethylation, coupled with selected reaction monitoring (dimethyl-SRM). We first demonstrate close to complete isotope incorporation for all peptides tested. The accuracy, reproducibility, and linear dynamic range of quantitation are further assessed based on known ratios of nonhuman standard proteins spiked into human cerebrospinal fluid (CSF) as a model complex matrix. Quantitation reproducibility below 20% (CV < 20%) was obtained for analyte concentrations present at a dynamic range of 4 orders of magnitude lower than that of the background proteins. An error of less than 15% was observed when measuring the abundance of 44 out of 45 major human plasma proteins. Dimethyl-SRM was further examined by comparing the relative quantitation of eight proteins in human CSF with the relative quantitation obtained using synthetic heavy peptides coupled to stable isotope dilution-SRM (SID-SRM). Comparison between the two methods reveals that the correlation between dimethyl-SRM and SID-SRM is within 0.3-33% variation, demonstrating the accuracy of relative quantitation using dimethyl-SRM. Dimethyl labeling coupled with SRM provides a fast, convenient, and cost-effective alternative for relative quantitation of a large number of candidate proteins/peptides.