MORFOLOŠKA ANALIZA NASTANKA I DIFERENCIJACIJE NEURONA U STANIČNOJ KULTURI, TIJEKOM RAZVOJA ZAMETKA I NAKON TRANSPLANTACIJE U MOZAK MIŠA UPORABOM MATIČNIH STANICA DOBIVENIH IZ MIŠJEG SOJA THY1 YFP-16

doctoral thesis

• Title (english): MORPHOLOGICAL ANALYSIS OF DEVELOPMENT AND DIFFERENTIATION OF NEURONS IN CELL CULTURE, DURING EMBRYONIC DEVELOPMENT AND AFTER TRANSPLANTATION INTO MOUSE BRAIN USING STEM CELLS DERIVED FROM MOUSE STRAIN THY1 YFP
• Author: Ivan Alić
• Mentor: Dinko Mitrečić (mentor)
• Mentor: Vesna Gjurčević-Kantura (komentor)
• Committee member: Dinko Mitrečić (predsjednik povjerenstva)
• Committee member: Vesna Gjurčević-Kantura (član povjerenstva)
• Granter: University of Zagreb; Faculty of Veterinary Medicine; Zagreb
• Defense date and country: 2021, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Veterinary Medicine; Basic and Preclinical Veterinary Sciences
• Universal decimal classification (UDC): 59 - Zoology

Abstract

Ishemijski moždani udar je najčešći uzrok trajnog invaliditeta u ljudi i drugi uzrok smrti u
razvijenim zemljama. Novu nadu u liječenje ove teške bolesti donose matične stanice koje
predstavljaju nediferenciranu populaciju stanica pomoću kojih se potencijalno mogu obnoviti
oštećeni neuroni. U ovom istraživanju je upotrijebljen mišji soj koji pod kontrolom promotora
Thy1 gena izražava žutu fluorescentnu bjelančevinu u cijelim neuronima, uključujući jezgru,
tijelo živčane stanice, akson, dendrite, te njihove trnove – spine. Koristeći izražaj bjelančevine
YFP praćen je nastanak i diferencijacija živčanih stanica u staničnoj kulturi i tijekom razvoja
živčanog sustava THY1 – YFP zametka, te su opažena zbivanja uspoređena s onima nakon
transplantacije živčanih matičnih stanica u mozak životinje kojoj je prethodno izazvan moždani
udar. Rezultati ovoga istraživanja su pokazali da je broj THY1 – YFP pozitivnih stanica tijekom
diferencijacije in vitro stalan, te se radi o 21,3–22,5% neurona prepoznatih pomoću biljega
MAP2, β3-tubulin i NeuN. Tijekom embrionalnog razvoja THY1 – YFP pozitivne stanice se
pojavljuju 12,5. dana razvoja zametka i njihov broj se povećava do kraja graviditeta u
središnjem, ali i u perifernom živčanom sustavu. Nakon trasplantacije u mozak životinje, THY1
– YFP stanice su se diferencirale, povezale sa stanicama domaćina i stvorile mreže u
ozlijeđenom tkivu. U kontrolnim životinjama stanice su se zadržale na mjestu transplantacije.
Ovo istraživanje je pokazalo da stanice koje izražavaju THY1 – YFP mogu biti korištene kao
jedna od metoda izbora u praćenju sudbine transplantiranih matičnih stanica, posebno u
istraživanjima koja zahtijevaju precizno praćenje stanične morfologije.

Abstract (english)

Ischemic stroke is the most common cause of permanent disability and the second leading cause
of death in developed countries. A new hope for treatment of this disease is brought by stem
cells which represent a specific cell population which can be potentially used to regenerate
damaged neurons. In this study we used the mouse strain which under the control of Thy1 gene
promoter expresses yellow fluorescent protein in all parts of neurons, including nucleus,
perikaryon, axon, dendrites and spines. By using a visible signal of YFP we described
emergence and differentiation of neurons in cell culture and during embryonic development.
These observations have been compared to processes taking place after stem cells were
transplanted into the brain of experimental animal affected by stroke.
MATERIAL AND METHODS
In this study mice embryo and adult mice from two mice strains, B6.Cg-Tg (Thy1-YFP) 16Jrs/J
(YFP-16) and C57BL/6NCrl, were used. For neural stem cell isolation pregnant females were
sacrificed on the 14.5th day of pregnancy, embryos were isolated and parts of the telencephalic
wall were dissociated and cells were placed in a specific proliferation medium. After formation
of neurospheres and their growth, they have been dissociated and placed on special substrates
containing poly-D-lysine and laminin to enable differentiation of neurons. After fixation
immunocytochemical staining was performed. Pregnant females were sacrificed for embryo
isolation from 9.5th to 17.5th day of pregnancy. Isolated embryos were fixed, cryosected and
prepared for immnohistochemical staining. Ischemic brain injury was induced by transient left
middle cerebral artery occlusion (MCAO) in adult C57Bl/6 mice. One day after MCAO,
stereotaxic transplantation was performed. Brains were isolated, cryosected and prepared for
immnohistochemical staining. Sections were analyzed and recorded using confocal microscope
Zeiss LSM 510 META.
RESULTS
Results of this study showed that the number of THY1 – YFP positive cells during
differentiation in vitro was constant and it was in the range from 21.3 to 22.5% of the total cell
count. THY1 – YFP positive cells were positive for SOX2 and all tested neuronal markers
(MAP2, β3-tubulin and NeuN). During embryonic development THY1 – YFP positive cells
were observed from E12.5 and their number has been constantly increasing in the central (spinal
cord, rhomencephalon and telencephalon) as well as in the peripheral (spinal nerves, cranial
nerves and retina) nervous system. THY1 - YFP positive cells during embryonic development
were positive for the same markers as during in vitro differentiation. In stroke affected mouse
brain transplanted THY1 – YFP cells differentiated and incorporated into the injured tissue. In
control animals the cells at the site of transplantation.
CONCLUSION
Results of this study showed that mature neurons as well as neuronal progenitor cells express
THY1 – YFP construct in in vitro conditions, during embryonic development and after
transplantation into the MCAO brain. This suggests that in addition to analyses of neuronal
differentiation in B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse, neural stem cells isolated from this
strain can be successfully used in studies where tracing of cells with neuronal fate is needed.
Key words: ischemic stroke, stem cells, differentiation potential, THY1, YFP

• Keywords: ishemijski moždani udar; matične stanice; diferencijacijski potencijal; THY1; YFP
• Keywords (english): ischemic stroke; stem cells; differentiation potential; THY1; YFP
• Language: croatian
• URN:NBN: urn:nbn:hr:178:710004
• Promotion: 2021
• Study programme: Title: doctoral study of anatomy, histology and embryology in veterinary medicine; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti (doktor/doktorica znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-03-24 10:33:33