(625c) Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells

Differential
Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced
Pluripotent Stem Cells

Julie Bejoy¹, Zhe
Wang², Brent Bijonowski¹, Qing-Xiang Amy Sang^2,3,
Teng Ma¹, Yan Li^*,1,3

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

3
Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida,
USA

Introduction:
A lack of well-established animal
models that can efficiently represent human brain pathology has led to the
development of human induced pluripotent stem cell (hiPSC)-derived brain
tissues. Brain organoids have enhanced our ability to understand the developing
human brain and brain disorders (e.g., Schizophrenia, microcephaly), but the
organoids still do not accurately recapitulate the anatomical organization of
the human brain. Therefore, it is important to evaluate and optimize induction
and signaling factors in order to engineer the next generation of brain
organoids. In this study, the impact of hyaluronic acid (HA), a major brain
extracellular matrix (ECM) component that interacts with cells through
ligand-binding receptors, on the patterning of brain organoids from hiPSCs was
evaluated. To mediate HA-binding capacity of signaling molecules, heparin was
added in addition to HA or conjugated to HA to form hydrogels (with two
different moduli). The neural cortical spheroids derived from hiPSCs were
treated with HA or heparin plus HA (Hep-HA) and were analyzed for ECM impacts
on neural patterning. The results indicate that Hep-HA has a caudalizing effect
on hiPSC-derived neural spheroids, in particularly for stiff Hep-HA hydrogels.
Wnt and Hippo/Yes-associated protein (YAP) signaling was modulated (using Wnt
inhibitor IWP4 or actin disruption agent Cytochalasin D respectively) to
understand the underlying mechanism. IWP4 and cytochalasin D were found to promote
forebrain identity. The results from this study should enhance the
understanding of influence of biomimetic ECM factors for brain organoid
generation.

Results
and Discussion: The results indicate that
the Hep-HA group favors ectoderm differentiation of hiPSCs compared to HA
alone. The cortical spheroids treated with different conditions were analyzed
for brain region-identity markers. The Hep-HA treatment increased the
expression of hindbrain markers HOXB4 and ISL1 compared to HA alone and gelatin.
The cells were also treated with ECM at an earlier stage of cortical tissue
development. The results indicated that Hep-HA effects on the neural patterning
depended on development stages (e.g., day 8 and 16). The cells were treated
with Wnt inhibitor IWP4 together with the ECM for evaluating the impact of Wnt
signaling on neural patterning. The IWP4 treatment abolished the effects of
Hep-HA treated cells, indicating that the cells in the Hep-HA group may express
elevated Wnt signaling. Hydrogel study with various modulus revealed that
Hep-HA hydrogels of higher modulus supported the hindbrain fate during hiPSC
neural patterning whereas the hydrogels of lower modulus supported forebrain
marker expression.

Conclusions:
This study investigates the
synergistic effects of heparin and hyaluronic acid on neural patterning of
hiPSC-derived cortical spheroids. The treatment of cortical spheroids with the
Hep-HA at late stage biases the neural patterning toward hindbrain fate. The
detailed evaluation of the underlying effects of different ECM reveals that
Hep-HA elevates Wnt signaling compared to HA alone. Modulation of the Hippo/Yap
signaling also influences the patterning effect of these ECMs. A comparative
study with Hep-HA hydrogels with varying modulus reveals that the lower modulus
(300-400 Pa) supports the forebrain fate whereas higher modulus (1000-1300 Pa)
supports hindbrain fate. Taken together, the Hep-HA hydrogels can interfere
with the cell signaling inside the cells and thereby influence neural
patterning of hiPSCs.

References: (1)
Colombres, M., Henriquez, J.P., Reig, G.F., Scheu, J., Calderon, R., Alvarez,
A., Brandan, E., Inestrosa, N.C., (2008) Heparin activates Wnt signaling for
neuronal morphogenesis. J Cell Physiology 216, 805-815.  

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