Poster Presentation 16
Derivation of Cortical Spheroids from Human Induced Pluripotent
Stem Cells in a Suspension Bioreactor
Yuanwei Yan, Liqing Song, Yan Li
Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University,
Tallahassee, Florida, USA
Abstract:
Human induced pluripotent stem cells (hiPSCs) emerge as a promising source to construct human brain-like
tissue in vitro for disease modeling and drug screening. While a suspension bioreactor can be used to generate
large size of brain organoids from hPSCs through enhanced diffusion, the influence of a dynamic bioreactor
culture environment on the brain tissue development from hiPSCs has not been well understood. The objective
of this study is to assess the effect of a suspension bioreactor on the cortical spheroid (i.e., forebrain-like
organoids) formation from hiPSCs. The bioreactor was seeded either with single undifferentiated hiPSK3 cells
or pre-formed embryoid bodies that were induced toward neural lineages. Aggregate size distribution, neural
marker expression (e.g., Nestin, PAX6, HOXB4, and beta-tubulin III), and cortical tissue patterning markers
(e.g., TBR1, BRN2, SATB2, and vGlut1) were evaluated in comparison to static culture control. Bioreactor
culture was found to promote the expression of TBR1, a deep cortical layer VI marker, and accelerated the
expression of SATB2, a superficial cortical layer II-IV marker that appears later according to “inside-out”
development pattern during cortical tissue generation. Prolonged culture after 71 days showed layer-specific
cortical structure in the spheroids. Differential gene expression of matrix metalloproteinase (MMP)-2 and -3
was also observed for bioreactor culture and static culture. The altered expression of cortical markers by a
suspension bioreactor indicates the importance of culture environment on the cortical tissue development from
hiPSCs.
Figure 1. A: The derivation
schematic paradigm of cortical
spheroid in the bioreactor. B:
Representative phase contract
images of spheroid at day 27
(scale bar 400 μm) and
fluorescent images of cortical
markers (Glut, GABA, TBR1,
SATB2, and BRN2) and synaptic
activity-related markers (SYN)
(scale bar: 100 μm). C: The gene
expression of neural markers by
RT-PCR
(day
32).
D:
Quantitative analysis of neurons
(β-tubulin III) and cortical
superficial
layer
marker
(SATB2) (day 30).
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