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tablished before stereotactic injection of control or green fluorescent
protein (GFP) expressing hNSCs into the PBBI penumbra one week later.
Animals were sacrificed at defined time points post-transplantation.
Brains were sectioned and assessed for GFP fluorescence.
Results:
The cells at week 1 post transplantation were undiffer-
entiated and morphologically indistinguishable from undifferentiated
cells
in vitro
. By week 5, a robust transplant could be seen sending out
processes as far as 2–3 millimeters. By week 8 the number of pro-
cesses was greater and they could be observed in the hind brain
(
*
12 mm from transplant site). The GFP processes emanating from
the transplant appear to follow intact white matter tracts in PBBI
brain. The processes were found to cross internal capsule in ipsilateral
hemisphere and traverse corpus callosum into contralateral hemi-
sphere.
Conclusion:
PBBI is conducive to human fetal neural stem cells
engraftment and survival. The transplanted cells appear to be capable
of sending processes over long distances.
Support:
This work was supported by State of Florida funding.
Keywords: Penetrating traumatic brain injury, Neural stem cell
transplants, FDA, Neurogenesis, Light sheet microscopy
D3-02
TRANSPLANTATION OF HUMAN INDUCIBLE PLUR-
IPOTENT STEM CELL-DERIVED NEURAL STEM CELLS
PROMOTES LOCOMOTOR RECOVERY AFTER SCI
Qi Lin Cao
, Yiyan Zheng, QIn Wang, shenglan li, ying liu, dong kim,
Qi LIn Cao
UT Medical School at Houston, Department of Neurosurgery, Hous-
ton, USA
Transplantation of hiPSC-derived neural stem cells (NSCs) could be
one of the most promised novel reparative strategies to promote
functional recovery after spinal cord injury (SCI). One of the major
challenges to fully realized the full therapeutic potential of hiPSC is
to purify the desired NSCs from differentiating hiPSCs in vitro be-
fore transplantation. In this study, we use the neural stem cell spe-
cific hiPSC reporter line and fluorescence-activated cell sorting
(FACS) to purify NSCs and then test its therapeutic potential for
SCI. The nestin-EGFP reporter hiPSCs, in which EGFP cassette has
been inserted to the nestin locus of hiPSC via homologous recom-
bination, are induced for neural differentiation and GFP expressing
NSCs are purified by FACS. The purified cells express NSC but not
iPSC markers. Importantly, the purified NSCs continue to proliferate
for a long time in vitro and differentiate into neurons, astrocytes and
oligodendrocytes under respective differentiation conditions. NOD-
SCID mice receive moderate contusion at T9 and then grafts of
hiPSC-derived NSCs, human fibroblasts or control medium at 8 days
after SCI. Robust survival of grafted NSCs is observed in all animals
receiving grafts at 2 months after transplantation. Some grafted
NSCs differentiate into NeuN
+
mature neurons and more into dou-
blecortin
+
immature neurons. Astrocyte or oligodendrcoyte differ-
entiation is also observed in grafted NSCs. The volumes of spared
white and gray are significantly increased in animals received NSC
graft. Furthermore, recovery of hindlimb locomotor function is
significantly enhanced in animals receiving grafted of hiPSC-derived
NSCs. No teratoma formation is observed in any animals receiving
hiPSC-derived NSCs. Our results show that the multipotential NSCs
can be purified from NSC specific reporter hiPSC line by FACS.
Importantly, purified NSCs can survive and differentiate into both
neurons and glias and promote functional recovery after transplan-
tation following traumatic SCI. These results suggest that hiPSC-
derived NSCs have great therapeutic potential for SCI and other
neurological diseases.
Keywords: spinal cord injury, neural stem cell, hiPSC
D4 Poster Session VII - Group D: Transplantation
D4-01
INTRACEREBROVENTRICULAR TRANSPLANTATION OF
ADULT NEURAL STEM CELLS (NSCS) AFTER TBI: PROOF-
OF-CONCEPT FOR ACTIVATION OF HOST NSCS
Regina Armstrong
, Genevieve Sullivan
USUHS, Anatomy, Physiology & Genetics, Bethesda, USA
Transplantation of neural stem cells (NSCs) may promote brain repair
by replacing lost cells and by interacting with the host tissue to
modulate the immune response and stimulate endogenous regenera-
tive capacity. Determining an effective NSC delivery route in the CNS
is particularly challenging for TBI that involves diffuse rather than
focal lesions. In clinical management of TBI, ventriculostomy is often
performed in patients with acute TBI after failure to control intra-
cranial pressure by other means. Ventriculostomy may provide a route
of access to the lateral ventricle for therapeutic delivery of stem cells.
The current studies provide a proof-of-concept test of in-
tracerebroventricular (ICV) delivery of adult NSCs into the lateral
ventricle in an impact-acceleration TBI model with traumatic axonal
injury in the corpus callosum. We also test the effect of NSC trans-
plantation in stimulating a regenerative response in endogenous NSCs
in the host subventricular zone (SVZ) based on activation of Sonic
hedgehog (Shh) signaling. Shh maintains the NSC niche in adult CNS.
Gli1 transcription indicates active Shh signaling. TBI from impact to
the skull at bregma was produced in adult
Gli1CreERT2
mice crossed
to
RosaTdTomato
reporter mice. NSCs were isolated from adult
UBI-
GFP
mice, which ubiquitously express green fluorescent protein
(GFP), and microinjected unilaterally into the lateral ventricle of the
Gli1CreERT2;RosaTdTomato
mice 2 weeks after the TBI or sham
procedure.
Gli1CreERT2;RosaTdTomato
host mice were then ad-
ministered tamoxifen to label endogenous cells responding to Shh
signaling after ICV transplantation. Mice were sacrificed for tissue
analysis at 4 weeks post-TBI. Transplanted GFP-NSCs survived,
maintained an immature phenotype, and were localized along the
ependymal lining of the ventricle and adhering to the choroid plexus.
Endogenous NSCs in the adjacent SVZ were fate-labeled with
TdTomato, indicating active Shh signaling, but were not changed
significantly due to the injury or ICV transplantation of GFP-NSCs.
An inflammatory response to transplanted GFP-NSCs was not ob-
served in sham or TBI mice. Funded by the DoD in the Center for
Neuroscience and Regenerative Medicine (CNRM).
Keywords: Transplantation, neural stem cell, Sonic hedgehog
D4-02
DIFFERENTIATION OF FDA-APPROVED HUMAN NEURAL
STEM CELLS WITH FUNCTIONAL IMPROVEMENT AFTER
A PENETRATING TBI
Aminul Ahmed
1
, Shyam Gajavelli
1
, Markus Spurlock
1
, Lai Yee
Leung
2
, Deborah Shear
2
, Frank Tortella
2
, Ross Bullock
1
1
University of Miami, Miami Project to Cure Paralysis, Miami,
United States
A-106