eBook: Advanced Tools Transforming Neurology Research - 16

neurons, astrocytes, and oligodendrocytes using
each respective known marker. At day 36, organoid
margins were positive for neural stem cell marker
NESTIN with high expression levels of TUJ1, a neuronal
cell marker (Figure 1A). On day 119, higher expression
levels of MAP2 revealed a significant presence
of mature neurons along with the presence
of oligodendrocytes visualized by OLIG2 markers
(Figure 1B, C). 32-day-old and 46-day-old cerebral
organoids also revealed rosette-like structures indicating
neuronal or ependymal cell differentiation
throughout the organoid. (Figure 1D, E).
Preliminary evaluation of cardiac organoids was
performed after reaching the maintenance stage,
11 days after embryoid body formation. After
reaching maturity, contraction activity could be visually
observed through light microscopy. The cardiac
organoid model was also positive for cardiomyocyte
marker cardiac Troponin (cTnT), revealing
the presence of cardiomyocytes throughout the
organoid body (Figure 2).
Figure 2. Immunostaining of cardiac organoids. (A) Magnified
view (20x) of cardiac organoids with cardiomyocytes were visualized
using the cTnT marker with nuclei visualized by DAPI. (B)
Presence of cardiomyocytes throughout the entire organoid can
be observed.
Organoid composition and
function evaluation
Figure 1. Staining of cerebral organoids for characterization. (A)
Immunostaining of cerebral organoid at day 36 for neural stem
cell marker NESTIN (red) and neuron TUJ1 (green) markers with
nuclei visualized by a DAPI counterstain. Neural stemness remains
near the borders of the organoid as shown by the NESTIN
marker. (B) Day 119 of cerebral organoid shows presence of oligodendrocyte,
shown by OLIG2 (pink) markers alongside neuron
TUJ1 (green) markers. (C) Mature neuron marker (MAP2)
dominates the borders as the organoid reaches its maintenance
stage. (D, E) H&E stain of a 32-day-old organoid section reveals
rosette-like structures within the 3D organoid structure.
RNA sequencing for known gene markers
Cerebral organoid composition was also evaluated
using bulk RNA sequencing for each organoid model
(Figure 3). Gene expression data was taken for cerebral
organoids at day 13, 44, and 100. Immature
cell markers such as NES and PAX6 decreased as the
organoids grew, revealing maturation and differentiation
of neural stem cells. Similarly, MAP2, DLG4/
PSD95 and SYP markers for mature cells increased.
Overall, cell diversity also increased, revealed by
positive markers highlighting an increase in expression
of astrocyte, oligodendrocytes, and other
neuron subclass markers.
Electrical impulse organoid function
Cardiac organoids were evaluated separately due
to the visual observation of contraction activity,
closely mimicking the diastole and systole phases
of cardiac conduction. A silicon probe with multichannel
reading capabilities was placed into the
organoid to capture the impulse generation and
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