Browsing by Subject "human embryonic stem cells"
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(2022)Puberty initiation is a crucial physiological process in human development. A group of hypothalamic neurons secreting the gonadotropin-releasing hormone (GnRH) and expressing the kisspeptin receptor (KISS1R) plays a key role in launching puberty. Furthermore, cellular KISS1R signaling has been shown to regulate GnRH expression and secretion. Although the in vitro differentiation of human pluripotent stem cells into GnRH-secreting neurons has been successful, it is of high interest to generate KISS1R expressing GnRH neurons. By utilizing the CRISPR activation technology, this study aimed to establish a conditional KISS1R-activation cell line using H9 human embryonic stem cells. Through controlling dCas9VP192 abundance using the Tet-On system combined with the dihydrofolate reductase destabilizing domain, the transcriptional activation of KISS1R was temporally regulated by the addition of two antibiotic drugs - doxycycline and trimethoprim. KISS1R expression was primarily assessed by qPCR and verified by immunocytochemistry and the use of a KISS1R-GFP reporter cell line. The main finding of this study is the achievement of a 6217 ± 2286 fold change in KISS1R transcription by introducing two guide RNAs (N = 3). Nevertheless, leaky gene activation was observed without drug treatment (fold change of 63 ± 51). Concludingly, this study successfully led to the generation of a KISS1R-activation cell line. After further characterization and refinement of the activation protocol, the established cell line will enable to investigate whether KISS1R upregulation modulates in vitro GnRH neuron differentiation, electrophysiology, hormone expression, and secretion in the future. Respective outcomes may lead to advances in understanding and treating pubertal disorders.
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MANF-Knockout Human Embryonic Stem Cells React to Oxidative Stress Differently than Wild-Type Cells (2023)Introduction: Oxidative stress occurs in cells when reactive oxygen species are generated as a by-product of oxygen metabolism and start to accumulate excessively. While extensive oxidative stress is highly detrimental to the cells, trophic factors help them survive. Trophic factor MANF has interested especially Parkinson’s disease researchers, but recent findings suggest that MANF plays a role in many diseases, also ones with an early childhood-onset. For this reason, it is important to investigate MANF function in different cell types. We have studied how MANF-knockout human embryonic stem cells react to oxidative stress compared to wild-type human embryonic stem cells, by exposing the cells to hydrogen peroxide and ethanol. Results: MANF-knockout human embryonic stem cells were more sensitive to oxidative stress than wild-type cells, but the variation between measurements was remarkable and the differences were statistically insignificant. We found that a transcription factor of our interest localized in the cell nuclei of MANF-knockout cells upon oxidative stress exposure. Such a nuclear translocation did not occur in wild-type cells. Moreover, we found that high concentrations (>2%) of ethanol reduced the viability of cells in only four hours. Discussion: Our findings suggest that MANF-knockout human embryonic stem cells react to oxidative stress differently than wild-type cells. Additional studies are necessary to clarify whether MANF-knockout human embryonic stem cells are indeed more sensitive to oxidative stress than wild-type cells. In the future, it would be interesting to inspect whether MANF protects human embryonic stem cells when the cells are exposed to physiologically relevant ethanol concentrations for longer periods of time.
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(2023)Rare mutations in the primate specific ZNF808 gene are a novel cause of pancreatic agenesis, a congenital developmental disorder that leads to neonatal diabetes. ZNF808 loss-of-function has been shown to lead to aberrant activation of regulatory MER11 elements, followed by upregulation of genes in proximity to these elements and increased expression of hepatic lineage markers. These findings suggest ZNF808 to play a key role in balancing the differentiation of endoderm progenitor cells between pancreatic and liver lineages during early human development. This thesis work aimed to study the gene regulatory mechanisms of ZNF808 in the differentiating endoderm progenitor cells to understand its function in controlling pancreatic lineage specification. This was achieved by comparing the lineage specification processes in wild-type (H1) and ZNF808 knockout (H1-ZNF808-KO) human embryonic stem cells (hESCs) during pancreatic differentiation. Further characterization of cellular heterogeneity and gene expression profiles upon ZNF808 loss was done using single-cell RNA sequencing (scRNA-seq). To validate the role of ZNF808 as the mediator of the observed lineage specification bias, the phenotype rescue was examined in a ZNF808 knockout overexpression cell line (H1-ZNF808-KO-OX). The results of this study demonstrate a clear lineage specification bias in the ZNF808 knockout, seen as divergence of the multipotent endoderm progenitors towards alternate hepatic and biliary fates at the posterior foregut stage. By modifying the pancreatic differentiation protocol, we were able to observe phenotype manifestation and cellular heterogeneity suppressed in the standard differentiation conditions. The scRNA-seq data analysis revealed the emergence of a biliary cell population showing upregulation of several hepatic markers, suggesting an alternative lineage specification process governed by ZNF808. Additionally, preliminary results from ZNF808 overexpression showed rescue of the ZNF808 knockout phenotype, further supporting its critical role in the normal pancreatic lineage development. In conclusion, these findings demonstrate the important role of ZNF808 in early human pancreatic development and warrant further studies on the detailed gene regulatory network guiding pancreatic lineage specification.
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