DOI: 10.6084/m9.figshare.4733257.v1
title: Supplementary Material for: Dedifferentiated Fat Cells as a Novel Source for Cell Therapy to Target Neonatal Hypoxic-Ischemic Encephalopathy

description: 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.