(521c) Functionalization of Brain Region-Specific Spheroids with Isogenic Microglia-like Cells

Functionalization
of Brain Region-specific Spheroids with Isogenic Microglia-like Cells

Liqing Song¹, Xuegang Yuan¹,
Zachary Jones², Cynthia Vied³, Yu Miao¹, Mark
Marzano¹, Jingjiao Guan¹,

Teng Ma¹, Yi Zhou²,
Yan Li^{1, *}

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

²Department of Biomedical Sciences, College of
Medicine, Florida State University, Tallahassee, Florida, USA

³The Translational Science Laboratory, College of
Medicine, Florida State University, Tallahassee, Florida, USA

Introduction:
Brain spheroids or organoids derived
from human induced pluripotent stem cells (hiPSCs) have shown great potential
for studying neurological disease progression and ZIKV virus infections. 
However, most cortical spheroids or organoids lack a microglia component, the
resident immune cells in the brain [1].  The objective of this study is to engineer
brain-region-specific spheroids from hiPSCs incorporated with isogenic
microglia-like cells in order to enhance brain-like microenvironments and
neural protective properties.

Materials
and Methods: Neural differentiation
was induced using dual inhibition of SMAD signaling with LDN193189 and SB431542
of human iPSK3 cells in low-attachment 24-well plates. Then the tissue
patterning was tuned through different patterning factors. Dorsal spheroids
were induced using the fibroblast growth factor-2 and ventral spheroids were
generated using IWP4 (a Wnt inhibitor) and purmophamine (Purmo, a sonic
hedgehog signaling activator). Microglial-like cells were derived from the same
hiPSC line using stage-wise growth factor induction into mesoderm (Fig.1).
Different microglia-specific markers (CD11b, IBA-1,
CX3CR1, and P2RY12) were characterized at different differentiation
stages.  The CellTracker Green labeled microglia-like cells were incorporated
into the dorsal/ventral spheroids to investigate the differential immune response
of co-cultured spheroids under pro-inflammatory stimulation.

Results
and Discussion: The derived microglia-like cells expressed several
phenotypic markers, including CD11b (70±6%), IBA-1 (80±5%), CX3CR1 (51±10%),
and P2RY12 (68±12%), and phagocytosed micron-size super-paramagnetic iron oxide
particles.  Microglia-like cells were able to upregulate pro-inflammatory genes
(TNF-α) and secrete anti-inflammatory cytokines (i.e., VEGF, TGF-β1,
and PGE2) when stimulated with amyloid β42 oligomers, lipopolysaccharides,
or dexamethasone.  Dorsal cortical (higher expression of TBR1 and PAX6) and
ventral (higher expression of Nkx2.1 and Prox-1) spheroids and organoids were
derived from the same hiPSC line, which displayed action potentials and
synaptic activities.  Co-culturing the isogenic microglia-like cells
with hiPSC-derived dorsal and ventral organoids showed differential migration
ability, Ca2+ transients imaging, and the response to pro-inflammatory stimuli
(ventral group had higher TNF-α expression).  The co-culture also
stimulated cell proliferation (higher BrdU+ cells) and reduced reactive oxygen
species expression,

Conclusions: The
microglia-like cells can be derived from hiPSCs and integrated with
brain-region specific organoids.  The co-cultured brain organoids better
resemble tissue-specific microenvironment and should advance our understanding
of the effects of microglia on brain tissue function.

References: [1] Haenseler W, Sansom SN, Buchrieser J, Newey SE, Moore CS,
Nicholls FJ, et al. A highly efficient human pluripotent stem cell microglia
model displays a neuronal-co-culture-specific expression profile and
inflammatory response. Stem cell reports. 2017;8:1727-42.

This study was supported by NSF CAREER
(1652992) and NIH R03EB020770.