Browsing by Subject "fibroblast"
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(2016)Heart failure is a major public health problem and a leading cause of mortality worldwide. The most common cause of heart failure is myocardial infarction. Following a myocardial infarction, a large number of cardiomyocytes die and cardiac muscle is replaced by fibrotic scar tissue. Since the adult heart has inadequate endogenous regenerative capacity, loss of muscle tissue often causes a progressive decrease in cardiac function eventually leading to heart failure. At the moment heart transplantation is the only curative treatment for heart failure, but the low number of donor hearts is limiting the use of this treatment option. As current drugs only slow down the progression of the disease, there is a great need for new regenerative treatments. Direct cardiac reprogramming is a new approach for generating cardiomyocytes for cardiac regeneration. Unlike pluripotent stem cell-based strategies, direct reprogramming enables conversion of a terminally differentiated cell type directly into another cell type without first producing a pluripotent intermediate. Due to their abundancy and role in the repair of myocardial injury, fibroblasts represent an attractive starting cell type for direct cardiac reprogramming. Fibroblasts have been directly reprogrammed to induced cardiomyocytes (iCMs) by overexpression of key cardiac transcription factors, microRNAs (miRNA) or by modulating specific signal transduction pathways with small-molecule compounds. Despite successful reports of direct reprogramming both in vitro and in vivo, the efficiency of direct reprogramming remains, however, too low for potential clinical applications. The aim of this M.Sc. thesis work was to establish direct reprogramming of mouse embryonic fibroblasts (MEFs) to iCMs by viral overexpression of cardiac transcription factors Hand2 (H), Nkx2.5 (N) Gata4 (G), Mef2c (M) and Tbx5 (T) and a small-molecule compound screening platform for identifying small-molecule compounds that could enhance the reprogramming efficiency and potentially replace cardiac transcription factors in direct cardiac reprogramming. In accordance with previous publications MEFs were successfully directly reprogrammed to iCMs using both HGMT and HNGMT cardiac transcription factor combinations. The screening platform was tested using the TGF-β inhibitor SB431542, which has recently been reported to increase the cardiac reprogramming efficiency. In line with previous publications, the reprogramming efficiency was significantly increased by treatment with SB431542. Initial tests with other small-molecule compounds did not have a positive effect on the reprogramming efficiency. The results of this M.Sc. thesis work verify previous publications and demonstrate a method for in vitro small-molecule compound screening, which can be used to identify compounds that increase the reprogramming efficiency in direct cardiac reprogramming. However, the results shown here are only preliminary and more replicates are needed in order to confirm the current results. Nonetheless, the results of this thesis work set a foundation for finding small-molecule compounds that in the future might be used to target direct cardiac reprogramming as a regenerative therapy for myocardial infarction and heart failure.
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(2023)Biallelic germline mutations in ERCC6L2 cause bone marrow failure (BMF) and predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The patients often develop varying cytopenias, and underlying hypoplasia in the bone marrow is usually evident. The aim of this thesis was to characterize the transcriptome of patient -derived fibroblasts with biallelic germline ERCC6L2 mutation. Moreover, the aim was to study changes on the gene expression at the RNA level in fibroblasts in different media conditions, ROS levels in ERCC6L2 -mutated fibroblasts, and whether addition of glutamine impacts the ROS levels. Fibroblasts from 16 skin biopsies were cultured; eight samples were from healthy controls and eight samples from patients with known mutations in ERCC6L2. Fibroblasts were cultured in different media conditions, followed by RNA extraction and RNA sequencing. We observed downregulation in base excision repair, nucleotide excision repair, mismatch repair, DNA replication, homologous recombination, and cell cycle in ERCC6L2 -mutated cells. MAPK signaling pathway, p53 signaling pathway, apoptosis, AMPK signaling pathway, and TGF-beta signaling pathway were in turn upregulated in ERCC6L2 -mutated cells. The medium did not affect the gene expression significantly across samples. We suspect that the effect of medium was not detected at the RNA level, but it might affect post-translational modifications. We also detected increased ROS levels in ERCC6L2 samples compared to control and observed decreased ROS levels in ERCC6L2 and control samples with excess glutamine. This study shows that biallelic mutations in ERCC6L2 do not only affect the bone marrow but can also affect tissues outside of the hematopoietic system. The transcriptomic analysis identified important biological processes, which could be studied with more detail in the future to further explore the pathology of the ERCC6L2 disease.
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(2022)The gastrointestinal (GI) epithelium is composed of a single layer of cells with a turnover time of only a few days. Due to its location at the barrier between GI tract contents and the underlaying mucosa, the epithelium is constantly exposed to stress such as toxic agents and a variety of pathogens and susceptible to injury. Accordingly, the homeostatic growth as well as repair of injury in epithelium must be efficient and strictly regulated. Misregulated repair of the injured epithelium can lead to pathologies such as chronic inflammation or cancer. Underlying stromal cells such as fibroblasts provide growth factors and other signaling molecules regulating the epithelial cell stemness, differentiation and repair, but the stromal regulatory pathways during regeneration are poorly understood. The aim of this study was to establish a consensus view on the heterogeneity of GI fibroblasts, as well as to map potential epithelium derived signals affecting fibroblast function in homeostatic and injury situations using literature review, in silico approaches, and murine primary intestinal fibroblast culture. Seurat and CellChat R packages were used to perform integration and interaction analyses of six previously published mouse and three human single- cell RNA-sequencing datasets of colonic epithelial and mesenchymal cells isolated in homeostatic and/or inflammatory conditions. Murine primary intestinal fibroblasts were treated with identified potential signaling factors ex vivo and 3’RNAseq was performed to identify transcriptional responses. Both mesenchymal and epithelial cell clusters were identified in the scRNAseq data. Interestingly, similar fibroblast populations could be found in the murine and human data. I identified several epithelium-derived signaling molecules potentially targeting GI fibroblasts and focused on Gas6-Axl pathway and lactate. I confirmed high and specific expression of the Gas6 receptor Axl in intestinal fibroblasts, but recombinant Gas6 failed to induce significant changes in cultured primary fibroblasts. Lactate-treated primary intestinal fibroblasts reprogrammed their transcriptome with main alterations in metabolic pathways and induction of neutrophil-attracting chemokines. In this work I suggest a consensus model for GI fibroblast subpopulations and suggest epithelium derived lactate as a powerful means to reprogram fibroblasts.
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