Assay for LIVE imaging of neuronal cell differentiation - Max

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Assay for LIVE imaging of neuronal cell
File no.: MI 0802-4674-MSG-ZE
Max-Planck-Innovation GmbH
Amalienstr. 33
80799 Munich
Phone: +49 (89) 29 09 19 - 0
+49 (89) 29 09 19 - 99
[email protected]
Dr. Matthias Stein-Gerlach
Tel.: 089 / 290919-18
[email protected]
Stem cell implantation has become an important therapeutic approach for organ regeneration, including
regeneration of brain functions after neurodegenerative diseases, but also after stroke. To elaborate
optimal conditions for functional improvement it is substantial to understand the mechanisms of the stem
cell graft in the host brain. So far invasive investigations are necessary, characterizing the stem cell fate
on tissue sections by immunohistochemistry. Hereby information on stem cell migration and
differentiation status or neuronal integration is obtained for one single time point after grafting. However,
as regeneration is a slow, chronic process, a full temporal profile of the mechanisms of action is needed;
up to today this is reached indirectly by resorting to group analysis, using standardized animal models
and relying on group average values which might not be fully reproducible.
There is an urgent need for a technology that takes into account the inter-individual, biological variability,
which can influence the experimental results substantially.
A series of neural stem cells, both human and murine, have been generated to express imaging reporters
of cell-specific gene activity during differentiation. Various stages of neuronal or glial differentiation are
selectively detectable during LIVE differentiation. Application of such transgenic cell lines serves in vivo
development of preclinical, stem cell based regenerative therapy strategies.
Also toxicology/drug screening of substances on neural cell types can be assessed during various
complete phases of differentiation with our tools. One-time-point ICC is replaced by continuous
monitoring of drug effect on cell dynamics in culture. Moreover, the positively screened drugs can be
further tested in vivo by noninvasive monitoring of the transgenic cell lines for longterm effects.
The dual reporter concept includes two lentiviral plasmids designed for constitutive and cell-specific expression
of bioluminescence and fluorescence reporters. The imaging reporter sets are selected to discriminate between
undifferentiated and differentiated state. Glial differentiation is monitored quantitatively by the GFAP promoter-driven Luc2, normalized to the constitutive
hRluc bioluminescence. The set of imaging reporters identifies unambiguously undifferentiated
(copGFP+/mCherry-) from differentiated (copGFP+/mCherry+) cells – verified by immunostainings for GFAPpositive astrocytes.
The dual reporter concept without additional normalization to the number of viable cells is suitable to visualize
neuronal differentiation. The time profile of Luc2 bioluminescence and copGFP fluorescence indicates a
progressive induction of doublecortin (DCX) promoter activity referring to an ongoing neuronal maturation.
Spontaneous differentiation of human NSCs into DCX-positive neurons is visualized by longitudinal in vivo cell
tracking with bioluminescence imaging (left panel). Cell proliferation is excluded in a separate set of animals
(right panel) receiving human NSCs with constitutively expressed Luc2.
We are looking for a licensing partner for this know-how generated by Mathias Hoehn, Annette
Tennstaedt and Markus Aswendt, from the in-vivo-NMR Laboratory, Max Planck Institute for
Neurological Research, Cologne.
Hoehn et al., Human neural stem cell intracerebral grafts show spontaneous early neuronal differentiation
after several weeks. Biomaterials 44 (2015) 143-154.