1. Naoki Tajiri1, Yuji Kaneko1, Kazutaka Shinozuka1, Hiroto Ishikawa1 et.al Plos One, 8(9)Sept 2013
Sandeep Satapathy
Roll-10079
Stem Cell Recruitment of Newly
Formed Host Cells via a Successful
Seduction?
Filling the Gap between
Neurogenic Niche and
Injured Brain Site

2. Exploring the Neurogenic Niche-
• The Sub-Ventricular Zone
(SVZ) and the Sub
Granular Zone /Dentate
Gyrus (DG) of the
Hippocampus of the
brain – active sites of
Neuronal Stem
Cells(NSCs) in the brain.
• However, in ischemic
conditions the cortical
region of brain has been
shown to be actively
involved in formation of
new neurons from these
NSCs.
"Neural Stem Cells - New Perspectives”, DOI:
10.5772/55426

3. Hypothesis:
Probably the cortex in a post injury condition, is facilitated to receive the
NSCs from the active stem niche like , SVZ and DG of hippocampus, in
order to compensate for the neuronal damage to regain the impaired
function??
If, this happens then; how does this migration takes place in the
brain???provided there are other cerebral tissues, which might pose to be
potential barrier in this long distance migration of NSCs.
Is there any other mediating pathway or factor that acts as a bridge or the
so called BIOBRIDGE , for this seduction of NSCs to happen????
Does the migratory pathway also manifest certain Extra cellular interacting
molecules, which in a concerted process is allowing this migration to
occur.

4. Ramification of Ideas:
Classically stem cells were looked as
• Source of Cell Replacement
• Bystander effects trophic factor secretion at localized tissues.
However, in present times, Stem cells have made a leap
• Into translational biology and applications of regenerative medicines with
approaches of modulation the Extracellular matrix biology.

5. Traffic Regulator:
Intracerebral Transplanted
Stem cells


Features:
• High level of extracellular metalloproteinases following a Intracerebral Stem
Cell implantation. The engrafted stem cell had a very short persistence
time.***
• Decreases host cell death and proliferation and formation of new host cells.
• Endogenous repair pathway onset, post a stem cell bio bridge formation
and regain of impaired motor and neurological functions.

6. Why low persistence time of engrafts not an
issue??
Prior to the start of the preliminary experiments, it was expected that the
Intracerebral infused MSCs would have very less retention potential at the site of
peri cortical lesions or the migratory exodus path from the SVZ/DG to the Cortex in
ischemic rats.
This was because of Hatting Mt, et.al(2009) who proved that in very short time
period, around a 20-30 days post MSCs infusion the stem cells permeate into the
pulmonary vasculature and start circulating in the arterial blood system.
This effect was termed as “PULMONARY FIRST PASS EFFECT”.
Findings:
Post 1 month graft survival: 0.6%
Post 3 months graft survival :0.16%

7. Procedures:
1. Control Animal Parameterization:
Prior to impacted brain injury, the animals were checked for baseline motor and
neurological functions and this was classified in terms of,
• EBST: Elevated body swing test,10 degrees shift from the central bodyline in a
hanging condition.
• Rotorod Maintenance Potential: The time period upto which the rats could keep
themselves stable on a fixed frequency rotating rod-Motor Coordination Index.
• Bederson’s Neurological Score: Based on the observable phenotypes like front
limb retraction post lateral displacement of 2-3 cms, beam walking ability under
a focused beam. These were scored from 0 sec (immediate response) to 3 sec
(delayed response)

8. 2. Traumatic Brain Injury Surgery (TBI):
The anesthetized rat animal models ,
underwent craniectomy and using a
precise metal impactor, the cortical
region of the brain was given a uniform
impact injury.
3.Graft Procedures:
The grafting of the messencymal stem cells (MSCs) were done at Intracerebral
locations(median cortex region and peri-cortex lesions) , days 7 post TBI, under
anesthetized conditions of the rat model. Around 105 cells were grafted for 3 ul per
deposit and three rounds were repeated of the same. The grafting was done post
checking of cross reactivity with monoclonal human specific antibody (HuNu).Animals
were left at 37°C for recovery and survival.
4.Cell Survival Analyses: Using high power field microscope, from a cryo preserved
section of the cortical region of the brain, the number of surviving host cells were
calculated in the peri cortex lesion area.

9. Histology:
1.Analyses of brain damage :
This was done staining the 4 coronal tissue sections of the dissected brain
using Hemotoxylin(H) & Eosin (E) stain. Together with this data and from the
calculated date of cell survivability in peri-cortical lesion area, the span of
tissue damage and the new host cell proliferation was accounted for post stem
cell transplantation. Using Abercrombie's formula the calculation of per
section was summed up for the entire brain.
2.Additional Immunohistochemical Analyses: Apart from analyzing the host
cell survivability and brain damage, to substantiate for the stem cell
bridge the following Immunohistochemical markers were also analyzed:
• For Host cell proliferation : Ki67 Marker
• Migration of NSCs from SVZ/DG to Peri cortex/cortex: Double cortin /DCX
marker.
• Immature neural marker :Nestin

10. Zymography:
To study the manifestation of unregulated
levels of extracellular metalloproteinases (MMP-9) ,
Zymography was done with gelatin based SDS PAGE. The
density of the bands forming clear strands against the
darkly stained undigested gel (stained using coomassie
brilliant blue) helps to quantify the expressional levels/
activity of a hydrolytic enzyme like MMP-9.
Recombinant enzyme was used as control strand in
each gel.
Autoradiography:
Because of the metals present in the enzyme posing for radioactive
properties, the tissue connecting the SVZ/DG to Cortex was cryodissected and
the membrane was blocked and further the MMP-9 was subjected to anti-MMP
9 antibody (mouse monoclonal),which later was amplified using goat anti mouse
antibody ,conjugated with Horse radish peroxidase for Immunofluorescence and
autoradiography assay.

11. Cell Migration Assay:
The Boyden chamber loaded with PRNCs (primary rat neuronal Cells),was
placed on a 24 well plate with confluent levels of MSCs (105 cells/well)
and starved with serum free DMEM/F-12 along with
Presence of cyclosporine A
Absence of cyclosporine A

12. Results:

13. Figure 1. Behavioral tests (performed by two investigators blinded to the treatment condition
throughout the study) were initially conducted at baseline (i.e., prior to brain insult) and
revealed that all adult SD rats included in this study displayed normal behaviors (A, B, and C).

14. Figure 2. The bio bridge between SVZ and impacted cortex consists of
highly proliferative, neutrally committed, and migratory cells.

15. Figure 3. At three months post-TBI, the brains from vehicle-infused animals displayed a
disparate pattern of cell fate in that the newly formed Ki67 positive and nestin labeled cells
were sequestered within the corpus callosum (A) and the SVZ (B) and only sporadic cells were
able to reach the impacted cortex (A’ and B’), with likely resident DCX cells seen around the
impacted cortex (C).

16. Figure 4. Laser-
captured
biobridge, correspondi
ng to the brain tissue
between SVZ and
impacted
cortex, expressed high
levels of MMP-9
gelatinolytic activities
at one month and
three months post-TBI
in animals
transplanted with
SB623 which were
significantly higher
than those TBI animals
that received vehicle
only or sham-operated
animals (*p’s< 0.05
vs. vehicle or sham;
Panel A).

17. Figure 5. After TBI, endogenous repair mechanisms commenced, but are limited to
the neurogenic SVZ and to a few quiescent resident neurogenic cells around the
impacted cortex (A).

18. Shortcomings:
Specific concentration to the median cortex area and peri lesion
cortex area, and thereby calculation of motor coordination index and
neurological score might give rise to certain false positives, as the
peripheral brain damage was ignored during baseline behavior
parameterization.

19. Future Possibilities:
• In most adult tissues, it is seen that the stem cell niche is kept aloof from
the active functioning cells and its recruitment follows a necessity of repair
and regeneration.So,in diseases where this recruitment process fails, can be
compensated for infused stem.
• Further the remodeling of host cells (into undifferentiated stem cells), at
the site connecting the stem cell niche and the zone of their functional
requirement ,can potential compensate for the need for external infusion
strategies.
• Migration of host cells from their stem cell niche to their zone of active
functional requirement, requires up regulation of MMPs and thus ECM
proteins like MMPs, can act as potential stroke therapies.
• Ultimately we can have something called “ STEM CELL VACCINES”

20. Take Home:
Stem cells may serve as bio bridges expressing MMP
profiles, that recapitulate the neurovascular unit
abetting the transplant mediated host cell migration
towards injured brain areas in affording functional
recovery in TBI.

21. References:
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PubMed: 17135412.
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