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Browsing by study line "Regenerative medicine"

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  • Nestaite, Ernesta (2023)
    Intestinal epithelium is capable of rapid regeneration, which is associated with transient changes in cellular identity. Some of these changes involve an enrichment of fetal-like gene expression and simultaneous suppression of adult stem cell signature. Interestingly, the upregulation of fetal-like marker Stem cell antigen 1 (Sca1) is modulated by extracellular matrix (ECM) which is known to guide epithelial cells during regeneration. Our recently published decellularized small intestinal ECM (iECM) system retains the composition and topology of natural ECM. This makes it an attractive system for ex vivo studies addressing regeneration. This thesis aimed to gain insight into the fetal-like identity and its dynamics using an ex vivo iECM system. Intriguingly, Sca1 expressing cells on iECM displayed migratory features, such as a leading edge and changes in nuclear morphology. Curiously, these features are typical for epithelial cells during development. Furthermore, based on marker gene expression during iECM re-epithelization, fetal-like state was upregulated while adult stem cell state was downregulated, revealing a gradually emerging inverse correlation. Additionally, data suggests that circadian rhythms may have a role in modulating the fetal-like state. iECM from an active-state mice indicated a reduced capability to induce fetal-like identity and overall re-epithelization compared to the rest-state iECM. The results of this thesis suggest further potential of iECM system in studying emergence of fetal-like state during re-epithelization and circadian rhythm impact on it.
  • Leppänen, Sonja (2023)
    IER3IP1 is a protein located in the endoplasmic reticulum (ER) transmembrane, and it is highly expressed in pancreatic beta cells and developing brain cortex. The loss-of-function mutations in IER3IP1 cause monogenic neonatal diabetes together with brain linked diseases such as epilepsy and microcephaly. The aim of this thesis is to study the role of IER3IP1 in the development and function of human beta cells using hESC-derived pancreatic islets. Using CRISPR/Cas9, IER3IP1 knockout (KO) and IER3IP1 loss-of-function mutation knock-in (KI) hESC clones were generated. For KO, the first exon of IER3IP1 was deleted whereas for KI, the 21. valine of IER3IP1 was changed to glycine. The clones together with their unedited controls (H1), were differentiated into pancreatic stem cell (SC)-islets following the optimized 7-stage differentiation protocol. The differentiation was followed during the protocol and the SC-islets were tested at the end of the protocol. In vitro, IER3IP1 KO-islets contained less beta cells and more alpha cells when compared to the H1-islets, as shown by immunostainings for insulin and glucagon. The beta cells of KO-islet accumulated more proinsulin compared to H1-islets and had significantly higher level of ER-stress shown by elevated ER-stress marker BiP. Moreover, the KO-islets showed drastically lower amount of insulin secretion and diminished insulin content. The IER3IP1 KI-islets did not significantly differ from H1-islets. Thus, this master’s thesis shows that IER3IP1 is essential for maintaining normal ER homeostasis and beta cell function in vitro. In future, these results should be confirmed using in vivo model.
  • Adrita, Chanda (2024)
    MELAS syndrome is a multi-organ disorder with a wide range of clinical manifestations, including hearing loss, cardiomyopathy, retinopathy, and stroke-like events. It can also be associated with type 2 diabetes. Eighty per cent of MELAS Syndrome cases have a 3243A>G mutation in the MTTL1 gene, which codes for tRNA (Leu-UUR). Although the exact cause of MELAS syndrome remains unknown, microvascular angiopathy and endothelial dysfunction, which result in reduced microvascular perfusion, are assumed to have a role in the reported phenotype. Yet, it is unclear how important this depicted angiopathy is in general and how it relates to the disease-specific stroke-like episodes in particular. In this thesis, I employed MELAS-patient-derived human induced pluripotent stem cell (hiPSC)-derived endothelial cells (EC) under flow as a disease model to investigate the possibility of an altered endothelial cell phenotype or function that could account for the disease phenotype. I used hiPSCs from healthy controls and patient-derived iPS-cell lines from MELAS patients with the 3243A>G mutation with different levels of heteroplasmy in all the experiments. Transcriptional patterns reflecting the flow response were observed, demonstrating that the MELAS hiPSC-EC cells respond to a flow stimulus. In the high-heteroplasmy cell lines, there was a significant downregulation of genes related to one carbon folate metabolism and serine biosynthesis, especially in response to flow, and these changes were also indicated in basal conditions. This is interesting as the same metabolic pathways have been shown dysregulated in other mitochondrial diseases. This study showed no appreciable difference in the mitochondrial oxygen respiration rate between MELAS-patient and control hiPSC-EC. These findings could link the disease phenotype to this web of pathways and explain the mechanism underlying the pathophysiology of MELAS syndrome.