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Browsing by study line "Regenerative medicine"

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  • Dynamics of fetal-like reparative state in the intestinal epithelium ex vivo 
    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.
  • The role of IER3IP1 in the development and function of human pancreatic beta cells 
    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.

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