Browsing by Subject "reprogramming"
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(2018)The capability to generate human induced pluripotent stem cells (iPSC) from somatic cells provides remarkable possibilities for regenerative medicine. However, prior to clinical applications the process of reprogramming should be optimized and carefully characterized. The purpose of this study was to get insight in reprogramming of human somatic cells to pluripotency using CRISPR-dCas9 activator system (CRISPRa). CRISPRa is a RNA guided bacterial nuclease system that has been modified for gene expression control. The study had two subprojects. The aims of the first subproject were 1) to reprogram hNESCs to pluripotency with CRISPRa in 2D culture, 2) to determine the efficacy of reprogramming and 3) to study whether CRISPRa-mediated pluripotent reprogramming pathway involves a mesendoderm-resembling intermediate state. The aim of the second subproject was to explore the possibility of CRISPRa-mediated endogenous gene activation and reprogramming to pluripotency also in 3D cell cultures. I performed the reprogramming in 2D and 3D cell cultures by using a dCas9 activator to induce different combinations of endogenous pluripotency reprogramming factors OCT4 (octamer-binding transcription factor 4), SOX2 (Sex determining region Y-box 2), NANOG, c-MYC, KLF4 (Krüppel-like factor 4) and LIN28. I analysed the results of the reprogramming at protein level, using alkaline phosphatase staining and immunocytochemistry, and at mRNA level, using qRT-PCR. The 2D reprogramming served as a proof-of-principle for reprogramming with CRISPRa. This study shows, that CRISPRa can be used to reprogram human neural stem cells to iPSC with different combinations of pluripotency reprogramming factors or by inducing a single master-regulator gene, OCT4. In addition, the reprogramming process was very efficient. I did not detect mesendodermal intermediate state in CRISPRa-mediated reprogramming to pluripotency, in contrast to published results from transgene- and small molecules-based reprogramming studies. Thus, this result suggests that the pathway leading to pluripotency differs between CRISPRa-mediated reprogramming and the two other reprogramming methods. CRISPRa can be used to initiate reprogramming also in 3D cell culture. However, in 3D cell culture the cells were not fully reprogrammed. Based on these findings, I postulate that CRISPRa serves as an alternative method for generating human iPSC. In addition, CRISPRa can be further developed into a platform for direct reprogramming of organoids for in vitro disease modelling in 3D.
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(2019)A novel method of somatic cell reprogramming employing CRISPR/Cas mediated gene activation (CRISPRa) may lead to improvements in the quality and efficiency of induced pluripotent stem cell (iPSC) generation by directly activating the endogenous factors of the cell. However, this method is yet to be optimized and is inefficient in its current form. Thorough characterization of the molecular events that occur during CRISPRa-mediated reprogramming could permit the fine-tuning of this method to improve iPSC production. B-lymphoblastoid cell lines (LCLs) adhere to culture plates during reprogramming, offering a form of selection for reprogramming cell populations. This thesis aimed to establish a system using LCLs for the characterisation of CRISPRa-mediated pluripotent reprogramming at the single-cell transcriptomic level. In this thesis LCL reprogramming conditions were characterized using alkaline phosphatase staining, immunocytochemistry, embryoid body formation, and live cell imaging. CRISPRa-mediated reprogramming efficiency was greatly increased by the targeting of the miR-302/367 cluster, a group of microRNAs known to improve the efficiency of transgenic reprogramming. Samples were collected for single-cell RNA sequencing (scRNA-seq) at multiple stages of reprogramming, the pluripotency of the iPSC samples was assessed, and a subset of the samples was sequenced. Clustering analysis of the sequencing data showed that the samples clustered apart from one another distinctly based on gene expression. The expression of notable genetic markers of LCLs, pluripotency, and developmental stages was consistent with the loss of somatic cell identity and rise of subpopulations characteristic of reprogramming. These results show that this is a functional system for scRNA-seq sample preparation that can be used to investigate reprogramming kinetics, and the samples collected will be part of a larger study of CRISPRa reprogramming.
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