EFFECT OF TRANSPLANTED NEURAL STEM CELL DOSE ON ENGRAFTMENT
EFFICIENCY, MIGRATION AND OLIGODENDROCYTE DIFFERENTATION IN SPINAL
CORD INJURED MICE
University of California, Irvine
1. Abstract
Human neural stem cells (hNSCs) are a potential for cell-based
regenerative therapy for spinal cord injury (SCI). Previously, we and several
other groups have shown that these cells can promote repair and recovery in a
variety of different models of central nervous system disease and injury. Our
research group has shown that hNSCs successfully integrate into host tissue
and enhance locomotor recovery in SCI rodent models (Cummings et al. 2005,
Hooshmand et al. 2009). In SCI host tissue, the majority of hNSCs
differentiated into oligodendrocytes that can myelinate neuronal processes
providing insulation for neuronal wiring that is essential for transmitting
electrochemical messages. Our previous data, along with other cell
transplantation studies, show that the majority of transplanted hNSCs
differentiate into oligodendrocytes, suggesting that remyelination may be the
primary mechanism for locomotor recovery after SCI (Tarasenko et al. 2007,
Karimi-Abdolrezaee et al. 2006, Keirstead et al. 2005, Liu et al. 2000,
McDonald et al. 1999).
The aim of this study is to investigate whether the cell dose of
transplanted human neural stem cells affects cell engraftment, migration, and
oligodendrocyte differentiation in a SCI rodent model. hNSCs were
transplanted at low, medium, high and very high doses (range from 10,000 to
500,000 cells) into spinal cords of immunodeficient NOD-scid mice 30 days
post injury (early-chronic phase) after moderate, 50kDy, mid thoracic
contusion injuries. Engraftment efficiency, cell differentiation, and migration
were analyzed from 30um thick coronal spinal cord sections after
immunohistochemistry using unbiased stereology (StereoInvestigator;
MicroBrightField Inc.). Our data suggests that hNSC engraftment efficiency
and number of human derived oligodendrocytes positively correlates with the
number of transplanted cells. Migration of hNSC does not seem to be
extensively affected by transplanted cell dose, but more hNSC derived cells
were found near the injection sites in both high and very high doses when
compared to low and medium doses. For further conclusion on possible
optimal transplantion cell dose for SCI therapy, the effect of transplanted
hNSCs doses on locomotor reccovery after SCI needs to be validated.
2. Introduction
SCI occurs after trauma to the spinal cord, causing compression or
bruising of the cord and loss of locomotor function, bladder control and
feeling below the site of injury. In the U.S., SCI currently affects more than
259,000 people with about 12,000 new cases per year. SCI is more
predominant among males, and the average age at injury is 40.2 years
(NSCISC). The extent of SCI ranges from complete injury to incomplete
injury; loss of locomotor and autonomic function ranges from complete to
incomplete, respectively (NSCISC). Individuals suffering from SCI can
undergo decompressive surgery to reduce the pressure on the spinal cord and
physical rehabilitative therapy, but few patients recover full locomotor ability
or full autonomic function below the site of injury (Fehlings, 2008).
Currently, there are no therapeutic treatments available for these individuals.
We are studying neural stem cell-based therapy as a possible treatment
for recovery from incomplete contusion-induced spinal cord injuries in murine
models. Previously, our research group has shown that hNSCs successfully
engraft, migrate away from the injury epicenter, and differentiate into neurons,
oligodendrocytes, or astrocytes following transplantation into spinal cords of
SCI mice. A large percentage of hNSCs differentiate into oligodendrocytes
when transplanted into host spinal cords 9 or 30 days after SCI, and the cells
are able to remyelinate host neuronal axons (Cummings et al., 2005,
unpublished data). Selective ablation of the transplanted hNSCs using
diptheria toxin led to loss of the observed locomotor recovery, suggesting that
the transplanted hCNS-SCs were responsible for the recovery observed in the
SCI mice (Cummings, et al., 2005). The aim of this study is to investigate the
relationship between hNSC dose, engraftment success, migration and
oligodendrocyte differentiation.
Table 1. Treatment groups ranged from low doses to very high
doses. The regular dose corresponds to the dose of stem cells
regularly administered in our lab.
Fig. 1. Traumatic spinal cord injury results in
both primary and secondary damage causing
loss of function below the level of epicenter
(Argosy Medical Animation).
Fig 4. The IH impactor used to administer
a spinal cord injury via a computer driven
probe, the force of the impact is set by the
person inflicting the injury.
Fig. 7. hCNS-SCs are injected bilaterally
at four different sites, two rostral and two
caudal to the site of injury. Doses
injected in a total volume of one
microliter per cord.
Gabriella Funes
Fig. 2. Image of hNSCs labeled with human-
specific cytoplasmic marker SC121 (brown)
and nuclear hematoxylin marker (purple).
Fig. 5. Number of transplanted hNSCs and
estimated averages of engrafted SC121
positive cells per dose groups at 16 weeks
post transplantations (wpt). Error bars
represent the group mean ± standard errors.
Fig. 8. Estimate number of SC121 positive cells
at specific distances along the cord per cell dose
group at 16 wpt. hNSC were injected rostral and
caudal from injury epicenter at T9 level 30 days
post injury. Distance in micrometers represents
the section distance from the most rostral section.
Fig. 3. Image of hNSCs labeled with human
specific-cytoplasmic marker SC121 (brown)
and nuclear Olig2 marker (blue).
Fig. 6. Estimate number of engrafted hNSCs and
SC121/Olig2 human-derived oligodendrocytes per
cell dose groups at 16 wpt. Error bars represent the
group mean ± standard errors.
Fig. 9. Estimate number of SC121/Olig2
positive cells at specific distances along the cord
per cell dose group at 16 wpt. Distance in
micrometers represents the section distance from
the most rostral section.
3. Methods and Materials
Mice:
• Female nonobese diabetic/severe combined immmunodeficient mice
(NOD-scid)
• Arrived at 8 weeks of age and acclimated for 2 weeks
• Injury inflicted at 10 weeks
Injury:
• Bilateral 50 kDyne moderate injury at thoracic segment 9 (T9)
• Inflicted using an Infinite Horizon Impactor (Fig. 2)
- causes hind-limb and bladder deficits
• Received anesthesia during the injury infliction, and analgesics several
days after the injury was sustained
Stem Cell Therapy:
• Human neural stem cells (hNSCs)
- n = 72 animals transplanted 30 days post injury
- every transplant is injected into 4 sites around the site of injury
- 2 rostral and 2 caudal injections
• Treatment groups (Table 1)
• Mice were euthanized at 16 weeks post transplantation (wpt)
Immunohistochemistry:
• Spinal cords were cut into 30 µ m sections on a Cryojane system
(Instrumedics, Inc.)
• Sections were stained in intervals of 24 with antibody against SC121
alone or SC121 and Olig2 (Fig. 2 & 3)
- SC121 - human-specific cytoplasmic marker
-Hematoxylyin - nuclear counterstaining mouse
- Olig2 - nuclear oligodendrocyte marker
Unbiased Stereology
• Cell estimates were calculated using the Stereo Investigator program,
version 8.0 (MicroBrightField, Inc.)
4. Results & Conclusions
Unbiased stereological quantification of human-specific cytoplasmic
marker (SC121) positive cells in coronal sections of the spinal cords at 16 wpt
revealed that engraftment efficiency and cell survival have a positive
correlation with the number of transplanted cells (Fig. 5). hNSCs transplanted
in low to high doses showed moderate proliferation capacity, which can be
seen by the increase in the number of SC121 positive cells when compared to
the transplanted cell doses (Fig. 5). In the very high dose group, the average
number of engrafted cells was lower than the number of transplanted hNSCs,
suggesting that at high doses hNSCs are less prone to proliferate. Transplanted
hNSC dose does not seem to extensively affect migration of the cells rostral
and caudal to the injury, but more SC121 positive cells are found near the
injection sites in both high and very high cell dose groups when compared to
low and medium cell doses (Fig. 8).
The estimate number of SC121/Oligodendrocyte transcription factor 2
(Olig2) positive cells (Fig. 3) positively correlates with the estimate number of
transplanted and engrafted SC121 positive cell numbers (Fig. 2 & 6). The
distribution of human-derived Olig2 positive oligodendrocytes does not seem
to be affected by the transplanted hNSCs doses (Fig. 9). Contrary to the
number of total engrafted hNSC-derived cells, SC121/Olig2 positive cells are
more evenly distributed across the cord in all cell dose groups (Fig. 9). This
suggests that SC121 positive cells near the injection sites likely represent other
neural cell types in the high and very high dose groups. The effects of
transplantated hNSC doses on locomotor recovery and on differentiation of
other neural cell lineages after SCI need to be validated for further conclusions
on possible optimal transplantation cell dose for SCI therapy.
Acknowledgements
-UCI’s Minority Science Program, the MBRS NIH Grant # GM-55246, and Dr.’s Marlene de
la Cruz and Luis Mota-Bravo for funding and support.
-Katja Piltti, Ph.D., Aileen Anderson, Ph.D. and Brian Cummings, Ph.D. and the CDRF
Animal Core for their guidance, mentoring and support.