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Improving Human Pluripotent Reprogramming by Targeted Activation of the miR-302/367 Cluster Using CRISPRa

Show simple item record 2020-02-12T11:00:30Z 2020-02-12T11:00:30Z 2020-02-12
dc.title Improving Human Pluripotent Reprogramming by Targeted Activation of the miR-302/367 Cluster Using CRISPRa en
ethesis.faculty Lääketieteellinen tiedekunta fi
ethesis.faculty Faculty of Medicine en
ethesis.faculty Medicinska fakulteten sv
ethesis.faculty.URI Helsingin yliopisto fi University of Helsinki en Helsingfors universitet sv
dct.creator Stadelmann, Christian
dct.issued 2019
dct.language.ISO639-2 eng
dct.abstract Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by transgenically expressing the four transcription factors OCT4, SOX2, KLF4, and C-MYC. This technology has revolutionised the stem cell field, yet cellular reprogramming is still inefficient and slow. To become fully applicable in regenerative medicine, the robust generation of safe and high-quality iPSCs from patient samples is essential. Various methods and potent reprogramming factors have been described to date. Yet, none have been able to circumvent these limitations markedly. The recently published activator-mediated approach (CRISPRa) is considered to be more physiological compared to the forced transgenic expression as the cell’s own genes are activated. Here, guide RNAs (gRNAs) mediate sequence-specific recruitment of non-cutting Cas9 (dCas9) activator proteins to the promoter region. Unlike other methods, it holds great multiplexing capacity and can also target enhancer and non-coding sequences. CRISPRa reprogramming still needs to be optimised since its efficiency is low. Thus, we aimed at enhancing this aspect and the temporal kinetics by targeting the micro RNA (miRNA) clusters 302/367 and miR-371-373, which both have been described as powerful cell fate regulators. We demonstrate successful reprogramming by targeting the miR-302/367 promoter alongside OCT4, SOX2, KLF4, C-MYC, LIN28A, REX1, NANOG, and EEA-motifs with CRISPRa. Activating the miRNA cluster results in a 2.5 fold efficiency increase in human foreskin fibroblast (HFF) reprogramming compared to the published basal CRISPRa system, quantified by staining for alkaline phosphatase. In HFFs, the CRISPRa efficiency is now comparable to the commonly used transgenic approach. Aiming to clarify the molecular mechanisms of these results, we characterised the expression of direct and downstream targets of miR-302/367 at different time points throughout the reprogramming process. Furthermore, validated with immunocytochemical stainings, the generated bona fide iPSCs express pluripotency markers and spontaneously differentiate into the three germ-layers, both signs of high-quality iPSCs. Beyond that, we report that miR-302/367 activation appears to result in earlier iPSC colony formation resulting in faster proliferating stem cell colonies shown with live-cell imaging. Employing a conditionally stabilised activator construct, we further show that with miR-302/367 targeting, the dCas9 activator expression seems to be required for only a short time period, sufficient to induce pluripotency. At the end of the project, the miR-302/367 cluster targeting was optimised and the best-working gRNAs were selected for further studies, which when combined further increase the CRISPRa-induced expression of the miR-302/367 cluster markedly. All in all, this study demonstrates that non-coding genetic elements like the miR-302/367 cluster can be targeted with CRISPRa, and its targeting significantly improves the reprogramming efficiency. Implications of the study for regenerative medicine and future steps are discussed. en
dct.subject Cellular reprogramming
dct.subject CRISPR/Cas
dct.subject CRISPRa
dct.subject induced pluripotent stem cells
dct.subject iPSCs
dct.subject miRNAs
dct.subject micro RNAs
dct.subject pluripotency
dct.language en
ethesis.language English en
ethesis.language englanti fi
ethesis.language engelska sv
ethesis.supervisor Weltner, Jere
ethesis.supervisor Trokovic, Ras
ethesis.supervisor Otonkoski, Timo
ethesis.thesistype pro gradu -tutkielmat fi
ethesis.thesistype master's thesis en
ethesis.thesistype pro gradu-avhandlingar sv
dct.identifier.ethesis E-thesisID:3a2542a7-a070-499a-8ca8-c98b90c7ec14
ethesis.degreeprogram.URI none und
ethesis-internal.timestamp.reviewStep 2020-02-11 15:33:10:116
dct.identifier.urn URN:NBN:fi:hulib-202002121343
dc.type.dcmitype Text Stem Cell Research und Biomedical Research und
ethesis.facultystudyline Cross-disciplinary translational medicine fi
ethesis.facultystudyline Cross-disciplinary translational medicine en
ethesis.facultystudyline Cross-disciplinary translational medicine sv
ethesis.facultystudyline.URI und
ethesis.mastersdegreeprogram Translationaalisen lääketieteen maisteriohjelma (Translational Medicine) fi
ethesis.mastersdegreeprogram Master's Programme in Translational Medicine en
ethesis.mastersdegreeprogram Magisterprogrammet i translationell medicin sv
ethesis.mastersdegreeprogram.URI und

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