(736c) Genomics Analysis of Metabolic Pathways of Human Stem Cell-Derived Microglia-like Cells and the Integrated Cortical Spheroids

Genomics
Analysis of Metabolic Pathways of Human Stem Cell-Derived Microglia-like Cells
and the Integrated Cortical Spheroids

Julie Bejoy¹,
Liqing Song¹, Mark Marzano¹, Yan Li¹

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

Introduction:
Brain spheroids or organoids derived
from human pluripotent stem cells (hiPSCs) are still not capable of completely
recapitulating in vivo counterpart and one of the limitations is lack of
microglia. To add build-in immune function, co-culture of the dorsal forebrain
spheroids with isogenic microglia-like cells (D-MG) was performed in our study.
The three-dimensional D-MG spheroids were analyzed for the transcriptome and
compared with isogenic microglia-like cells (MG). Cortical spheroids containing
microglial-like cells displayed different metabolic programming, which may
affect the associated phenotype. The gene expression for glycolysis and hypoxia
signaling was increased in co-cultured D-MG spheroids, showing the metabolic
shift to aerobic glycolysis, which is in favor of M1 polarization of
microglia-like cells. In addition, the metabolic pathways and the signaling
involved in cell proliferation, cell death, PIK3/AKT/mTOR signaling, as well as
Wnt and Notch pathways were analyzed. The results showed the activation of mTOR
and p53 signaling, increased expression of Notch ligands, and the repression of
NF-kB and canonical Wnt pathways in the co-cultured D-MG spheroids, which are
consistent with the lower expression of cell cycle genes. These analyses indicate
that physiological 3-D microenvironment can reshape the immunity of in vitro
cortical spheroids and may better recapitulate in vivo brain tissue
function for disease modeling and drug screening.

Materials
and Methods: Neural differentiation
was induced using dual SMAD inhibitors LDN193189 (LDN) and SB431542 (SB) on
human iPSK3 cells in low attachment plates. The neural progenitor spheres were
treated with patterning factors cyclopamine (a sonic hedgehog inhibitor) and
fibroblast growth factor-2. The identity is defined by TBR1, PAX6, BRN2 and
SATB2. Microglial cells were generated by initiating mesoderm induction using
Activin A, BMP4, SCF and VEGF, followed by SCF, Flt3L, IL-3, and GM-CSF
treatment. Non-adherent cells were replated to tissue culture-treated plates in
the presence of IL-3 and M-CSF to derive glial cells. Dorsal spheroids (day 30)
were co-cultured with isogenic microglia-like cells at 4:1 ratio (8×10⁵
neurons to 2×10⁵ microglia-like cells) in 50% DMEM/10% FBS and 50%
neural differentiation medium composed of DMEM/F12 plus 2% B27. After three
days of co-culture (day 33), the dorsal spheroids containing microglia-like
cells (D-MG group) and the microglia-cells only (MG group) were harvested for
RNA-sequencing.

Results
and Discussion: The central metabolic
pathways were verified, and the values were shown as an increase in glycolytic
and pentose phosphate pathways in D-MG group as well as the increased amino
acid synthesis. Hypoxia is an important factor in affecting stem cell
metabolism and phenotype. Our results did not show higher HIF-1α gene
expression in the D-MG group but showed the increased expression of HIF-1α
pathway downstream genes. In order to investigate how metabolic reprogramming
is orchestrated, the status of critical pathways for cellular function,
including PI3K/AKT/mTOR, Myc, p53, nuclear factor kappa-B (NF-kB), Wnt, and
Notch, were analyzed. Our results showed that the expression of mTOR (0.66) was
slightly elevated in D-MG spheroids and the downstream effects were inhibited.
Also there was a reduction in Myc activity inside the D-MG spheroids indicating
the slow proliferation of cells. The canonical Wnt pathway is reduced in the
D-MG spheroids, whereas the non-canonical Wnt pathway was activated in the spheroids.
 Notch ligands were up-regulated in the D-MG spheroids.

Conclusions:
The D-MG spheroids had higher
expression of genes for glycolysis and hypoxia signaling, showing the metabolic
shift to aerobic glycolysis, consistent with M1polarization of microglia. Signaling
pathway activities (activation of mTOR and p53, repression of NF-kB and canonical
Wnt) are consistent with slower proliferation rate and the accumulation of differentiated
cells. The additional NADPH needed for citrate and lipid synthesis was mainly
generated by pentose phosphate pathway activation. The D-MG group enriched
genes for NOTCH signaling, but not canonical Wnt signaling. The MG group had
higher expression of genes for cell cycle and proliferation.

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Dranka, B.P.,Schwartz, B., DeBerardinis, R.J., (2016) Reductive carboxylation
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This study was supported
by NSF CAREER (1652992) and NIH R03EB020770.