| dc.date.accessioned |
2020-02-12T11:00:30Z |
|
| dc.date.available |
2020-02-12T11:00:30Z |
|
| dc.date.issued |
2020-02-12 |
|
| dc.identifier.uri |
http://hdl.handle.net/123456789/27211 |
|
| 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 |
http://data.hulib.helsinki.fi/id/a4d5aaa2-b5aa-41a7-ba4c-e5e0df7a902d |
|
| ethesis.university.URI |
http://data.hulib.helsinki.fi/id/50ae46d8-7ba9-4821-877c-c994c78b0d97 |
|
| ethesis.university |
Helsingin yliopisto |
fi |
| ethesis.university |
University of Helsinki |
en |
| ethesis.university |
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.URI |
http://data.hulib.helsinki.fi/id/languages/eng |
|
| 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 |
| ethesis.thesistype.URI |
http://data.hulib.helsinki.fi/id/thesistypes/mastersthesis |
|
| 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 |
|
| ethesis.discipline.med |
Stem Cell Research |
und |
| ethesis.discipline.med |
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 |
http://data.hulib.helsinki.fi/id/SH_TMED-510 |
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 |
http://data.hulib.helsinki.fi/id/MH30_002 |
und |
