Supplementary Material for: Dedifferentiated Fat Cells as a Novel Source for Cell Therapy to Target Neonatal Hypoxic-Ischemic Encephalopathy
Mikrogeorgiou A.
Sato Y.
Kondo T.
Hattori T.
Sugiyama Y.
Ito M.
Saito A.
Nakanishi K.
Tsuji M.
Kazama T.
Kano K.
Matsumoto T.
Hayakawa M.
10.6084/m9.figshare.4733257.v2
https://karger.figshare.com/articles/journal_contribution/Supplementary_Material_for_Dedifferentiated_Fat_Cells_as_a_Novel_Source_for_Cell_Therapy_to_Target_Neonatal_Hypoxic-Ischemic_Encephalopathy/4733257
<p>Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) remains a major
cause of mortality and persistent neurological disabilities in affected
individuals. At present, hypothermia is considered to be the only
applicable treatment option, although growing evidence suggests that
cell-based therapy might achieve better outcomes. Dedifferentiated fat
(DFAT) cells are derived from mature adipocytes via a dedifferentiation
strategy called ceiling culture. Their abundance and ready availability
might make them an ideal therapeutic tool for the treatment of HIE. In
the present study, we aimed to determine whether the outcome of HIE can
be improved by DFAT cell treatment. HI injury was achieved by ligating
the left common carotid artery in 7-day-old rat pups, followed by 1-h
exposure to 8% O<sub>2</sub>. Subsequently, the severity of damage was
assessed by diffusion-weighted magnetic resonance imaging to assign
animals to equivalent groups. 24 h after hypoxia, DFAT cells were
injected at 10<sup>5</sup> cells/pup into the right external jugular
vein. To evaluate brain damage in the acute phase, a group of animals
was sacrificed 48 h after the insult, and paraffin sections of the brain
were stained to assess several acute injury markers. In the chronic
phase, the behavioral outcome was measured by performing a series of
behavioral tests. From the 24th day of age, the sensorimotor function
was examined by evaluating the initial forepaw placement on a cylinder
wall and the latency to falling from a rotarod treadmill. The cognitive
function was tested with the novel object recognition (NOR) test. In
vitro conditioned medium (CM) prepared from cultured DFAT cells was
added at various concentrations to neuronal cell cultures, which were
then exposed to oxygen-glucose deprivation (OGD). The number of cells
that stained positive for the apoptosis marker active caspase-3
decreased by 73 and 52% in the hippocampus and temporal cortex areas of
the brain, respectively, in the DFAT-treated pups. Similarly, the
numbers of ED-1-positive cells (activated microglia) decreased by 66 and
44%, respectively, in the same areas in the DFAT-treated group. The
number of cells positive for the oxidative stress marker
4-hydroxyl-2-nonenal decreased by 68 and 50% in the hippocampus and the
parietal cortex areas, respectively, in the DFAT-treated group. The HI
insult led to a motor deficit according to the rotarod treadmill and
cylinder test, where it significantly affected the vehicle group,
whereas no difference was confirmed between the DFAT and sham groups.
However, the NOR test indicated no significant differences between any
of the groups. DFAT treatment did not reduce the infarct volume, which
was confirmed immunohistochemically. According to in vitro experiments,
the cell death rates in the DFAT-CM-treated cells were significantly
lower than those in the controls when DFAT-CM was added 48 h prior to
OGD. The treatment effect of adding DFAT-CM 24 h prior to OGD was also
significant. Our results indicate that intravenous injection with DFAT
cells is effective for ameliorating HI brain injury, possibly via
paracrine effects.</p>
2017-07-18 09:56:52
Dedifferentiated fat cells
Brain injury
Conditioned medium
Rice-Vannucci model
Excitotoxicity
Neuroprotection
Oxidative stress
Behavioral tests