Page 362 - IJB-9-2

International Journal of Bioprinting Laser bioprinting of hiPSC-derived neural stem cells and neurons

tubulin (green, Figure 3A), which already stains neurons in most common ones among these lineages. Figure 3G shows
their earliest phases of differentiation, and mature neuronal vGLUT1 staining (green) for glutamatergic neurons and
marker MAP2 (green, Figure 3B and 3C). The left images of GABA-antibody staining (red) for GABAergic neurons
Figure 3A–C depict larger overviews of the printed pattern. of printed NSCs (same sample and image section for both
In all cases, there was a large proportion of proliferating staining) at day 30 post-differentiation (d32 pp, diff 0/30).
cells. However, many post-differentiated NSCs migrated The comparison of the panels indicates that most of the
and proliferated during neuronal differentiation until NSCs differentiated to glutamatergic neurons and a smaller
day 12. At day 12, post-differentiated NSCs proliferated proportion became GABAergic neurons, which is a common
everywhere. There is also strong migration of d20 neurons proportion in the cerebral cortex; ratios of approximately
but less proliferation until day 12. However, at day 12, these 70%–80% glutamatergic and 20%–30% GABAergic neurons
neurons proliferated almost exclusively at the position of have been reported by other groups [60,61] .
the printed droplets.
3.7. Differentiation of NSCs to glial cells under
3.5. Maintenance of multipotency neuronal differentiation conditions
The maintenance of multipotency of printed NSCs is Besides differentiation to neurons, NSCs are also
demonstrated in the panels of Figure S2 (in Supplementary capable to differentiate to glial cells, such as astrocytes
File). Stem cell marker nestin (green), PAX6 (red), SOX2 and oligodendrocytes. Such differentiation also occurs
(red) and β3-tubulin (green) are depicted together with spontaneously, even under culture conditions supporting
Hoechst 33342 cell nuclei staining (blue) and phase contrast neuronal differentiation. Hence, differentiation of printed
imaging at day 2 post-printing with different microscope NSCs to astrocytes (Figure 3E) and oligodendrocytes
magnifications. Obviously, all cells still expressed these (Figure 3F) under neuronal differentiation conditions was
stem cell markers at day 2. investigated.

3.6. Neuronal differentiation of NSCs and neurons The differentiation to astrocytes in neuronal
after printing differentiation medium is shown in Figure 3E with the
The composition of cell types and the ratio of neurons to markers GFAP (red), MAP2 (green), S100B (green, for
NSCs changes during culture under neuronal differentiation mature astrocytes), and Hoechst 33342 (blue) at day 2 (day
conditions. Figure 3D shows early-born cortical neuron 2 pp, diff 0/0), day 23 (day 23 pp, diff 0/21), and day 37
nuclei marker TBR1 staining (red) and MAP2 staining (day 37 pp, diff 0/35) post-printing. These staining reveal
(red) together with Hoechst 33342 cell nuclei staining that there are no GFAP-positive astrocytes at day 2 before
(blue) of printed NSCs and d5 neurons, respectively, starting neuronal differentiation. In contrast, there are
22 days after printing (day 22 pp, diff 0/20 for NSCs; day already many GFAP-positive astrocytes at day 23. However,
22 pp, diff 5/22 for d5 neurons). In the TBR1 images, the these cells did not increasingly appear at the position of the
position of four printed droplets can still be seen due to printed droplets but often in the interspace and mixed with
the blue Hoechst staining, while there is only one droplet neurons (MAP2-positive). At day 37, most astrocytes are
position depicted in MAP2 images due to higher resolution mature (S100B-positive).
microscopy. It can be seen that printed NSCs, which were Figure 3F shows staining with oligodendrocyte marker
under differentiation culture conditions, migrated and O4 and Hoechst 33342 at day 2 (day 2 pp, diff 0/0), day
populated the whole depicted region, although the highest 35 (day 35 pp, diff 0/33), and day 67 (day 67 pp, diff 0/65)
cell density remained at the droplet positions. Most of the post-printing. While we observed no O4-positive cells at
NSCs, post-differentiated for 20 days, were TBR1-positive day 2 before starting neuronal differentiation, there was
and about half of them were MAP2-positive, indicating already an abundance of O4-positive cells at day 35, most
an abundant presence of early-born cortical neurons. D5 of them were pre-oligodendrocytes (with simple dendritic
neurons showed less migration and proliferation. They extensions without bifurcations) with a few immature
were much more concentrated in the printed positions oligodendrocytes (with a few bifurcations only). At day
and arranged in ring-like formations. Less than 50% of d5 67, mature oligodendrocytes (with long extensions and
neurons were TBR1-positive 22 days after printing, but the complex bifurcations) could also be observed.
proportion was higher among migrated cells and lower
in the printed positions. This difference did not exist in 3.8. Impact of astrocytes on NSCs and neurons
MAP2 expression, which is generally much higher.
under neuronal differentiation
Further culturing under neuronal differentiation The effect of astrocytes, added to or spontaneously
conditions led to specific differentiation in different neuronal differentiated from NSCs, on NSCs and d20 neurons under
lineages; glutamatergic and GABAergic neurons are the neuronal culture conditions for 23 days was investigated and

Volume 9 Issue 2 (2023) 354 https://doi.org/10.18063/ijb.v9i2.672