(174by) Wnt-Notch Signaling Interactions during Neural and Glial Patterning of Human Induced Pluripotent Stem Cells

Wnt-Notch Signaling Interactions during Neural and Glial Patterning of
Human Induced Pluripotent Stem Cells

Julie Bejoy¹, Brent
Bijonowski¹, Teng Ma¹, Yan Li^1,2

1
Department of Chemical and Biomedical Engineering; FAMU-FSU College of
Engineering; Florida State University; Tallahassee, FL USA

2
Department of Chemistry and Biochemistry, Florida State University,
Tallahassee, Florida, USA

Introduction:
The human brain formation involves
complicated processing which is regulated by a gene regulatory network involving
different signaling pathways. The cross-regulatory interactions between
elements of different pathways affect the process of cell fate assignment
during neural and glial tissue patterning. In this study, the interactions
between Wnt and Notch pathways, the two major pathways that influence neural
and glial differentiation of human induced pluripotent stem cells (hiPSCs)
individually, were investigated. In particular, the synergistic effects of
Wnt-Notch pathway on the neural patterning processes along anterior-posterior
(A-P) or dorsal-ventral (D-V) axis of hiPSC-derived cortical spheroids were
explored. The human cortical spheroids derived from hiPSCs were treated with
Wnt activator CHIR99021 (CHIR), Wnt inhibitor IWP4, Notch inhibitor (DAPT)
individually, or in combinations (CHIR+DAPT, IWP4+DAPT).

Materials and Methods: Human iPSK3
cells were seeded into Ultra-Low Attachment (ULA) 24-well plates. The formed
EBs were treated with dual SMAD signaling inhibitors 10 μM SB431542 and
100 nM LDN193189. After 8 days, the cells were treated with fibroblast growth factor-2
and retinoic acid until day 16. During day 16-21, the spheroids were treated
with six different conditions: (1) control, no small molecule was used; (2)
CHIR99021 (10 μM), which activates Wnt signaling by inhibiting glycogen synthase
kinase-3β; (3) IWP4, which inhibits Wnt signaling through the porcupine
and thereby reducing the production of Wnt proteins; (4) the Notch inhibitor
DAPT. The γ-secretase inhibitor DAPT can inhibit the γ-secretase
dependent S3 cleavage of Notch, which releases the Notch internal cytoplasmic domain;
(5) CHIR99021 plus DAPT (C+D); (6) IWP4 plus DAPT (I+D). To elucidate
the influence of regional identity of neural progenitors on glial differentiation,
the spheroids (day 21) of the six conditions were switched to BMP-4 treatment
up to day 32.

Results
and Discussion: In our study,
combinational treatments of Wnt and Notch modulators affected brain regional
identity of iPSC-derived spheroids. The addition of DAPT to CHIR or IWP4
influences neural patterning effects of canonical Wnt pathway. Notch inhibition
with DAPT reduced the hindbrain marker HOXB4, but the CHIR treatment was able
to maintain HOXB4 expression in the presence of DAPT. Analysis of forebrain
markers TBR1 and FOXG1 showed that IWP4 cannot maintain their expression in the
presence of DAPT. I+D also reduced the expression of ventral forebrain marker Nkx2.1.
The cortical spheroids that were treated with Wnt and Notch signaling
modulators were then switched to gliogenesis by BMP4 treatment. The results showed
that the CHIR group expressed the higher levels of astrocyte markers GFAP and
Vimentin compared to the other groups. This might be because that CHIR
treatment increased Notch activity and Notch ligand expression in the derived
cortical spheroids. The lowest expression of astrocyte markers was shown in I+D
group, indicating the dominance of DAPT treatment over IWP4 treatment on
astrocyte differentiation and importance of Notch activation for astrogliogenesis.

Conclusions:
In this study, the functional
cross-talk between Wnt and Notch signaling on neural and glial tissue
patterning from hiPSCs were investigated using Wnt and Notch modulators. The
results suggested the possibility of repressing effects of Notch inhibitor DAPT
on Wnt inhibition and the positive impact of Wnt activation on Notch signaling
for brain regional identity of hiPSC-derived cortical spheroids. Notch
inhibition with DAPT reduced the hindbrain marker HOXB4, but CHIR was able to
maintain HOXB4 expression in the presence of DAPT. Analysis of forebrain
markers TBR1 and FOXG1 showed that IWP4 plus DAPT reduced forebrain marker
expression.  CHIR reduced the impact of DAPT on brain regional patterning
whereas DAPT reduced Wnt inhibition effects. By switching the derived cortical
spheroids to gliogenesis with BMP4, Wnt activator CHIR promoted the expression
of astrocyte markers GFAP and Vimentin. The addition of Notch inhibitor DAPT to
CHIR reduced astrocyte marker expression, confirming the importance of Notch
activation on astrocyte generation.

References Hayward,
P., Kalmar, T., Arias, A.M., (2008) Wnt/Notch signaling and information processing
during development. Development 135, 411-424.

This study was supported
by NSF CAREER (1652992) and NIH R03NS102640.