Establishing basic principles of optogenetics in a cell replacement therapy model of Parkinson’s disease : : the transduction of ventral mesencephalic dopaminergic progenitor cells

Abstract: The objective of this presented study was to establish and demonstrate the proof of principle of a protocol for the
transduction of dopaminergic progenitors from embryonic ventral mesencephalons in vitro and in vivo for the
application of optogenetics in a cell replacement therapy (CRT) rat model of Parkinson’s disease (PD). The
whole process comprised the review and determination of the transduction parameters, the implementation of the
elaborated and combined protocol of dissection, transduction and cell culturing or transplantation and the
analysis and evaluation of the impact of the transduction process on the cells’ composition and functionality.
It was predefined to perform the transduction with lentiviruses, carrying the EF1a as a strong ubiquitous
promotor, the WPRE as expression enhancement element, the EYFP as reporter gene and the ChR2(H134R) as
the designated opsin to be expressed, at a MOI of 0.5 for six hours stored in an Eppendorf tube, to wash the cells
afterwards to remove the virus and subsequently process the cells for either cell culture (in vitro) or
transplantation (in vivo). The cell cultures were fixed after ca. 2 weeks and the animals sacrificed after 4 weeks
as interval for expression. The analysis included a restriction digest, antibody test against EYFP and virus
titration as preparation for the transduction and ICC, cell counts and qPCR for the in vitro part and amphetamine
rotation tests, fIHC, IHC and cell counts for the in vivo part.
The restriction digest confirmed the integrity of the plasmid, the antibody test the suitability of the GFP-antibody
to detect the reporter gene and the virus titering the successful production of lentiviruses at a concentration of
106 copies / ml.
The assessment of the in vitro data showed that instantly the virus incubation and washing steps resulted in a
great general cell loss but barely impacted the cells’ viability. In the short term the storage in an Eppendorf tube
by itself also did not have a major negative impact on the cell composition. However, in the long term it did
reduce the growth potential of all neurons and the virus incubation that of neurons but not of dopaminergic
progenitors. Transduced cells were clearly identifiable in the cell cultures but the number of EYFP-positive
dopaminergic progenitors and therewith the transduction efficiency was lower than expected. The levels of
expression assessed by qPCR though were higher.
The in vivo data - even if limited in validity due to small sample size and variances within the results - showed a
similar decrease in the total cell number but not in the viability immediately after the incubation and washing.
The survival rates of dopaminergic progenitors in the transduced group were surprisingly higher than those in the
non-transduced control group and similar to normal rates from the literature. But the lesser changes in rotation
scores implied a possible impaired functionality. The survival rates of all transduced cells were low and even
lower than the analogous rates in vitro. Though the in vivo and in vitro parts are hardly comparable due to
different reasons. Transduced cells were clearly identifiable in brain slices within the transplants. There were
also EYFP-positive dopaminergic progenitors distinguishable yet rarely.
Overall, the established protocol proved as a safe and successful step-by-step procedure. The virus incubation by
itself did not have major negative impacts and most notably not on the number of dopaminergic neurons.
Additionally, the rate of expression was higher than the rate of transduction indicating a lack of quantity but not
of quality in the conduct. However, further investigations are required for technical improvements, efforts for a
greater efficiency e.g. by increased virus titers, MOIs and yields of dopaminergic progenitors and to answer the
question of possible impaired functionality for a feasible application of optogenetics in the cell replacement
therapy model of PD

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2018

Classification and Topics

• Classification: Medizin, Gesundheit
• Keyword: Optogenetik ; Parkinson-Krankheit ; Zelltransplantation ; Dopaminerge Nervenzelle ; Transduktion ; Vorläuferzelle

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2018
• Creator: Schlanstein, Paul Jakob
• Contributor: Coenen, Volker Arnd ; Albert-Ludwigs-Universität Freiburg. Medizinische Fakultät

Further information

• DOI: 10.6094/UNIFR/16239
• URN: urn:nbn:de:bsz:25-freidok-162398
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 14.08.2025, 10:45 AM CEST