(282d) Assembly of Human Stem Cell-Derived Vascular Spheroids and Cortical Spheroids to Model 3-D Brain-like Tissues

Song, L., Florida State University
Li, Y., Florida State University
Yuan, X., FAMU-FSU College of Engineering
Ma, T., FAMU-FSU College of Engineering

Assembly of Human
Stem Cell-Derived Vascular Spheroids and Cortical Spheroids to Model 3-D
Brain-like Tissues

Liqing Song, Xuegang Yuan, Teng Ma, Yan Li

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

Introduction: Human
cerebral spheroids derived from induced pluripotent stem cells (iPSCs) provide
novel tools for recapitulating the cytoarchitecture of the human brain and for
studying biological mechanisms of neurological disorders.  However, the
heterotypic interactions of neurovascular units, composed of neurons,
pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like
tissues are less investigated.  The objective of this study is to investigate the
impacts of neurovascular interactions on the brain regional patterning and
function of cortical spheroids and organoids derived from human iPSCs. 

Materials and Methods: Hybrid neurovascular spheroids were constructed by
fusion of human iPSC-derived cortical neural progenitor cell (NPC) spheroids,
endothelial cell (EC) spheroids, and the supporting human bone marrow mesenchymal
stem cells (MSCs). The neural differentiation of human iPSK3 cells was induced
using dual inhibition of SMAD signaling with LDN193189 and SB431542. Then the tissue
patterning was tuned through the treatment with retinoic acid and fibroblast
growth factor-2. Endothelial differentiation of hiPSK3 cells was induced
through Wnt activation by CHIR99021. Single hybrid spheroids were constructed
at different NPC: EC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in
low-attachment 96-well plates. Neural patterning markers, cell-cell
interactions, and ECM remodeling markers were compared among different hybrid

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Results and Discussion: The incorporation of MSCs
upregulated the secretion levels of VEGF-A, PGE2, and TGF-β1.  In
addition, tri-cultured spheroids promoted the expression of TBR1 (deep cortical
layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and
MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and
cell-cell communications on cortical tissue patterning.  Moreover, tri-culture
system elevated blood-brain barrier relevant genes (e.g., GLUT-1), CD3 and
tight junction protein ZO-1 expression.  By co-culturing multiple cell
types in the 3D spheroid system, brain-like tissues showing capillary
structures and interacting with neurons in the basement membrane were derived. 
Treatment with the CXCR4 antagonist, AMD3100, showed
the immobilization of MSCs during spheroid fusion.

Differential heterotypic cellular interactions promoted
cortical differentiation and layer separation by modulating the soluble factor
secretion, e.g. VEGF-A, and extracellular matrix remodeling. In addition,
co-cultured ECs exhibited the properties of brain microvascular cells.
Therefore, this forebrain-like model has potential applications in
understanding the cellular interplay of the neurovascular unit in the diseased
human brain and in screening the novel drugs before clinical trials.

[1] Song L, Tsai A-C, Yuan X, Bejoy J, Sart S, Ma T, Li Y.  Neural
differentiation of spheroids derived from human induced pluripotent stem
cells-mesenchymal stem cells co-culture.  Tissue Engineering Part A.  2018, In
Press. DOI:10.1089/ten.TEA.2017.0403.

study was supported by NSF CAREER (1652992) and NIH R03EB020770. 15D Engineering
the Tissue and Cell Microenvironment