At the Center for Life Nano Science (CLNS@Sapienza – Italian Institute of Technology) in Rome, Dr Silvia Di Angelantonio and her collaborator Dr Alessandro Soloperto are focusing their efforts on generating a robust protocol of differentiation for recapitulating the retinal neuron development in vitro, and thus investigate how alterations of TAU protein folding might be connected to retinal physiology and pathology.
In particular, in-house derived retinal ganglion cells (RGSs) were generated from a healthy induced-pluripotent stem cell (iPSC) line through a multi-step differentiation approach which relies on the use of small molecules in order to commit stem cells on the proper path of differentiation. The protocol lasts approximately 30-35 days, leading to a functional, homogeneous and well-distributed 2D network of retinal neurons. Then, cells were fixed and stained with DAPI for DNA (shown in white) and three differentiation markers to confirm the generation of a specific subtype of retinal neurons: the retina ganglion cell. Here the list of the markers used:
- MAP2 (yellow), a microtubule-associated protein present in the cytoskeleton of neuronal dendrites.
- Tuj1 (magenta), which stains the neuron-specific class III beta-tubulin, the major component of axonal cytoskeleton and is widely used as a marker to distinguish neurons from other cell types.
- Brn3a (cyan), this transcriptional factor is expressed in multiple sites in the central nervous system including the retina. Herein, Brn3a recognizes specific subtypes of retinal ganglion cells, regulating the expression of downstream genes involved in axon pathfinding, dendritic stratification and target selection.
Acquisitions have been performed with a Nikon Ti2 inverted microscope equipped with CrestOptics Confocal Spinning Disk X-Light V3, camera Photometrics 95B (11 um pixel size) and Celesta laser source (Lumencor). We show 40x images (Nikon 40x water objective, NA 1.25) obtained with a stitching tool (Figure A) and a 3D volume acquisition of a region of interest (Figure B).
The possibility to make a large view with an automatized acquisition system allows researchers to evaluate the degree of differentiation within the whole cell culture. In Figure A, Tuj1 staining (magenta) gives an overview of the neurite arborization pattern and, in combination with Brn3a staining (cyan), allows to match this information with the identification of retinal ganglion cells,which show Brn3a positive nuclei. Given that, it was possible to focus on a specific area in order to acquire a 3D Z-stack and, afterwards, perform quantitative analysis.
In Figure B, it is shown a maximum intensity projection and a volume view of the 3D Z-stack acquired in the region of interest. Moreover, measurements have been performed on a specific area shown in Figure B and an example is shown in Figure C. For instance, by thresholding DAPI (white) and Brn3a (cyan) positive cells and then combining the two thresholds obtained, it was possible to count how many objects are positive for both (e.g. 10 objects were measured as “combined threshold” – shown in orange – on a total count of 21 nuclei identified with DAPI; measurements have been obtained with NIS Element “General Analysis” tool). This data lead to calculate the efficiency of the differentiation protocol in the cell culture (i.e. 47%).
Noteworthy, human stem cell-derived retinal ganglion cells may represent not only a novel in vitro model to deepen neurodegenerative processes occurring in the brain and the retina during, for instance, aging and diseases such as Alzheimer and Parkinson, but also a model for testing therapeutic treatments.
Large View (stitching)
Maximum Intensity Projection
Count of Brn3A positive nuclei