Generation and characterization of a double reporter cell line for following maturation of hESC-derived β-cells

master's thesis

• Title (croatian): Uspostavljanje i karakterizacija stanične linije sa sustavom dva reportera kao modela za praćenje sazrijevanja beta-stanica izvedenih iz embrionalnih matičnih stanica
• Author: Vida Kufrin
• Mentor: Anthony Galavas (mentor)
• Mentor: Inga Urlić (sumentor)
• Committee member: Inga Urlić (predsjednik povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Domagoj Đikić (član povjerenstva)
• Committee member: Romana Gračan (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Diabetes mellitus is a group of metabolic disorders primarily affecting pancreatic β-cells. Due
to the limitations of existing therapy options for insulin-deficient diabetes, great efforts are put
into developing cell-based replacement therapies utilizing human pluripotent stem cells.
However, existing differentiation protocols, based on developmental principles in vivo, often
result in the generation of cells that are not fully mature and functional. To improve and
optimize existing protocols for hPSC reprogramming, we developed a novel H1 INSeGFP/
MAFA-mCherry double reporter cell line using CRISPR-Cas9 technology. Herein,
detailed characterization and assessment of differentiation potential of novel cell line is
reported. H1 INS-eGFP/MAFA-mCherry reporter line can reproducibly and efficiently
differentiate to form β-cells while preserving pancreatic identity likewise paternal unmodified
H1 cell line. Furthermore, H1 INS-eGFP/MAFA-mCherry derived β-cells express functionality
and maturation related genes and can respond to glucose challenges in vitro. Also, both GFP
and mCherry integrated reporter genes are fully functional in novel cell line. Reported findings
led to the validation of a novel reporter cell line that will serve as a valuable tool in unraveling
novel strategies to achieve a high-efficiency differentiation that presents one more step required
for the translation of this approach to the clinic setting.

Abstract (croatian)

Dijabetes je metabolički poremećaj koji primarno pogađa β-stanice gušterače. Budući da su
postojeće terapijske opcije za pacijente sa smanjenom razinom inzulina ograničene, velike nade
polažu se u stanične terapije temeljene na humanim pluripotentnim matičnim stanicama.
Međutim, postojeći protokoli diferencijacije, temeljeni na razvojnim principima in vivo, često
rezultiraju generacijom β-stanica koje nisu u potpunosti zrele i funkcionalne. Kako bi se
protokoli diferencijacije unaprijedili i optimizirali, razvili smo novu dvostruku reportersku
liniju H1 INS-eGFP/MAFA-mCherry koristeći tehnologiju CRISPR-Cas9. U ovom radu
prikazana je detaljna karakterizacija i procjena diferencijacijskog potencijala nove stanične
linije. Linija H1 INS-eGFP/MAFA-mCherry ima sposobnost efikasne i reproducibilne
diferencijacije u β-stanice pritom zadržavajući identitet usporediv s nemodificiranom
roditeljskom staničnom linijom H1. Nadalje, H1 INS-eGFP/MAFA-mCherry izvedene β-
stanice eksprimiraju gene važne za funkcionalnost i sazrijevanje te imaju sposobnost odgovora
na promjene u koncentraciji glukoze in vitro. Također, ugrađeni reporterski geni GFP i mCherry
su potpuno stabilni i funkcionalni u ovoj staničnoj liniji. Provedeno istraživanje potvrdilo je
ispravnost nove stanične linije koja će biti korištena kao vrijedan alat u dizajnu novih strategija
za efikasnu diferencijaciju te predstavljati jedan korak više ka kliničkim ispitivanjima novih
terapija za dijabetes.

• Keywords: pancreas; stem cells; hESC; beta cells; diabetes; differentiation; GFP
• Keywords (croatian): gušterača; matične stanice; hESC; beta stanice; dijabetes; diferencijacija; GFP
• Language: croatian
• URN:NBN: urn:nbn:hr:217:277341
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-09-28
• Created on: 2021-10-08 10:38:33