1) The study characterized host vascularization of transplanted human neural stem cell grafts in porcine spinal cords. Twelve grafts of human neural progenitor cells were transplanted into three pigs' spinal cords. 2) Six weeks after transplantation, the pigs were sacrificed and the grafts were analyzed. Graft survival ranged from 0-65% and inversely correlated with host microvascular density in the grafts. More vascularized grafts had less cell survival. 3) Vessels in the grafts expressed vascular endothelial growth factor and contained proliferating endothelial cells and immune cells, which may impact graft survival. The findings could influence future immunosuppression regimens for cell transplantation.

1. Characterization of Local Vascularization of Transplanted Human Neural Stem
Cell Grafts in the Porcine Spinal Cord
1,2Jason Lamanna, 1Victor Hurtig, 1Elman Amador, 1Juanmarco Gutierrez, 1Lindsey Urquia,
1Thais Federici, and 1,2Nicholas Boulis.
Background: Cell Transplantation in the Spinal Cord
• Clinical trials transplanting cell-based therapeutics into the spinal
cord are underway for a range of neurological diseases, including
Multiple Sclerosis (MS), Spinal Cord Injury (SCI), and Amyotrophic
Lateral Sclerosis (ALS)
• Direct intraparenchymal transplantation has been safely and
effectively utilized by our group in over 150 pigs and 27 clinical
trial subjects (Figure 1)
• Limited information is known about the post-transplantation fate
of cell grafts in clinical trials, including the immunological factors
that impact graft survival
• Immunosuppression regimens in these trials are adapted from
experience with solid organ transplantation
• In pre-clinical studies conducted by our group, we observed a
heterogeneous pattern of cell graft survival between animals and
between individual cell grafts in the same animal
• Understanding the post-transplantation fate of grafted cells is
essential to the widespread clinical translation of cell-based
therapeutics
Objective
The purpose of this study was to quantify host vascularization into transplanted human neural
progenitor cell grafts and correlate this with graft survival in a large animal spinal cord model.
Transplantation and Graft Identification
Funding and References
• Amyotrophic Lateral Sclerosis Association
• Regenerative Engineering and Medicine Center
References
• Klein et al. Human Gene Therapy, 2005
• Riley et al. Neurosurgery, 2009
Vascular Endothelial Growth Factor Expression
Conclusions
Characterization of Vascularization into Transplanted Cell Grafts
1Neurosurgery, Emory University, Atlanta, GA, United States; 2Biomedical Engineering, Emory University & Georgia Institute of Technology, Atlanta, GA, United States
Figure 1: Human intraspinal stem cell
transplantation. Stabilized injection
platform fixated to the spine of a human
clinical trial subject at Emory University.
Human neural stem cells were directly
injected in to the spinal cord (insert).
Transplantation
• Three female Gottingën minipigs underwent a multi-level laminectomy of the thoracolumbar spine,
opening of the dura mater, and placement of a spine-mounted stereotactic injection apparatus
• Twelve 2.5 x 105 cell grafts (25 μL) of human neural progenitor cells (provided by Clive Svendsen at
Cedars Sinai) were directly transplanted into the spinal cord parenchyma
Post-Operative Management
• Pigs were maintained for 6 weeks after transplantation with Tacrolimus (0.025 mg/kg BID IV)
• Daily assessment of sensory and motor function revealed transient, expected post-operative deficits
and full recovery to baseline for all animals within 7 days
Histological Analysis
• Pig were sacrificed 6 weeks after transplantation, spinal cords harvested following transcardiac
perfusion with 4% PFA, and serially sectioned at 50 μm
• Every 6th section was stained for the human nuclear antigen to identify cell grafts
• Stereological quantification of every 6th sectioning using uniform random sampling with the Cavalieri
principle and an optical dissector was performed to calculate individual graft survival
0
20
40
60
80
%Engraftment
200 μm 200 μm
Figure 2. Transplanted Graft Identification and Stereological Quantification. Representative micrographs of transplanted human
neural progenitor cell grafts in the porcine spinal cord with DAB-enhanced human nuclear antigen staining (mouse monoclonal
antibody, MAB1281) with cresyl violet counterstain. A representative non-rejected graft located at the grey/white mater junction with
limited inflammatory infiltrate and over 30% of cells surviving (A). A representative rejected graft from the same animal with less than
5% of cells surviving and a large inflammatory response adjacent to the non-rejected graft (B). The heterogeneity of cell survival of the
36 grafts is apparent with graft-wise stereological quantification (mean 22.0% cell engraftment, range of 0.0 - 65.7%) (C).
A B C
200 μm 200 μm
A
B
0 20 40 60 80
0
2
4
6
8
10
Engraftment %
MicrovascularDensity
r = -0.60, p = 0.0002
D
EC
F
Figure 3. Characterization of Host Vascularization into Transplanted Cell Grafts. The transplanted grafts were identified with human
nuclear antigen staining and cell survival quantified with stereology. Representative micrographs of non-rejected (A) and rejected (C)
grafts are shown. At the center of each graft, co-staining for CD31 (green, mouse monoclonal ab186720) and GFAP (red, rabbit
polyclonal ab7260) was performed. In the representative non-rejected grafts, few large vessels are apparent at low magnification (B).
However, in the rejected graft, numerous infiltrating vessels were apparent (D). Five high-powered fields (E) with the most vessels were
acquired for each graft and average micro-vascular density (MVD) was calculated (mean 18.3 infiltrating vessels per graft, range 3 – 46).
Linear regression showed a statistically significant inverse correlation (r = -0.60, p = 0.0002) between MVD and cell engraftment (F).
CD31 - GFAP - DAPI
• Inverse correlation between host vascularization of transplanted cell grafts and graft survival
• Infiltrating vessels are proliferating, express VEGF and contain numerous immune cells in the
perivascular space
• Studies underway to assess vascular infiltrates of transplanted cell grafts at different time points and
in an allograft model of cell transplantation
• These findings could impact future immunosuppression regimens employed in cell transplantation
Transmission Electron Microscopy and Endothelial Cell
Proliferation
BA Figure 4. Vascular Endothelial Growth Factor
Expression. At the center of transplanted cell grafts, co-
staining for CD31 (green) and VEGF (red, rabbit
polyclonal ab53465) was performed. In low
magnification micrographs, diffuse VEGF expression was
observed in the vessels (A). At higher magnification with
confocal microscopy, VEGF was observed in vascular
endothelial cells and in cells in the perivascular space,
such as T cells (B).
Figure 5. Further Characterization of Infiltrating Vessels.
Transmission Electron Microscopy of cell graft-containing tissue
showed immune cells in the perivascular space of small vessels
not detected with fluorescent microscopy (A, B). Confocal
microscopy of vessels stained for Ki67 (red, ab15580), a marker of
cell proliferation, showed numerous Ki67+ vascular endothelial
cells.
CBA