1. bFGF (100ng/ ml), EGF (20 ng/ ml)
Neuroprotective effects of intravenous injection of human embryonic stem cell-neural progenitor cells in traumatic optic neuropathy mouse model
Zahra Seiedrazizadeh1, Susan Simorgh1, Farzad Pakdel2, Mohammad Javan1, Hossein Baharvand1, Leila Satarian1,*
1. Department for Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
2. Ophthalmic Research Center, Tehran University of Medical Sciences, Tehran, Iran.
Objectives
Methods
Results
Retinal ganglion cells (RGCs) take signals coming from visual stimuli in the eye and any damage to the optic nerve would lead to death of irreplaceable RGCs.
Optic nerve damage can lead to a total loss of vision, a permanent organ damage that in most cases is not curable through surgery or medication.
Neuroprotective functions of stem cells in the nervous system have prompted many studies investigating the effectiveness of a range of these cells on various retinal disease models.
Neural progenitor cells (NPCs) secrete an assortment of trophic factors that are vital to the protection of the visual system.
Studying the effects of 25,000 NPCs intravenous injection through visual cliff showed any vision improvement in NPCs compared to the vehicle group. In contrast, retrograde tracing test and IHF staining of the RGCs revealed their significantly higher RGCs concentration in NPCs group. Moreover study of Retinal thickness showed more thickness in NPCs group compared to vehicle group. Most of the tests indicating that the NPCs protect RGCs from degeneration.
Three animal groups were used for comparison: 1) Healthy 2) Vehicle and 3) NPCs. These behavioral groups were compared using the Visual Cliff behavioral test, immunohistoflourscent staining using Brn3a marker for RGCs and GFAP for astrocytes, retrograde tracing with DiI injection into the superior colliculus, and study Retinal layer thickness using H&E staining. C
21 days post ONC
60 days post ONC B A B A
Intact
Vehicle
hESC- NPCs P0 P8
D12 D0 A
Noggin (100 ng/ ml)
Noggin (250 ng/ml)
RA (2 µM)
bFGF (100ng/ ml), EGF (20 ng/ ml) 6 12
days
200
200
200
Fig 3. Visual Behavioral test 60 Days after crush. A. Mouse in the visual cliff box, B. Vissual behavioral test on day 21, and C. day 60 showed any significant difference between NPCs and vehicle groups.
Fig 5. Studying RGCs survival between groups 60 days after crush A. Retrograde tracing, , show RGCs survival increases in NPs group than model group. and B. Hematoxilin & Eosin by Light microscopy for Intact, Vehicle, and NPCs group. B
N-CADHERIN
NESTIN A
Intact
Day 60
Day 21
200
Vehicle
hESC-NPCs
Conclusion
NCAM
SOX2
Due to ability of NPCs to secrete trophic factors, they may be used for optic nerve protection.
References
Fig 1: hESC-Neural progenitor cells generation and characterization. A. Light microscopy; Differentiation of hESCs to the NPCs. B. Immunocytofluorscent for N-Cadherin, NCAM, Nestin, and Sox2 as a neural progenitor markers.
1. Satarian et al, PLOS one, 2013.
2. Drago et al, Biochimie, 2013.
3. Aoki et al, Graefes Arch Clin Exp Ophthalmol, 2008. B
21 Days after ONC
60 Days after ONC
………
………. 2 4
60 days 6 21
Cell 25,000/ Media 200 μl; IV Injection
Tissue collection
ONC A ** ** B
ONC
Intact
Acknoledgement
Fig 4: Whole mount Retina staining with Brn3a, 21 and 60 days after Crush. B. quantitative difference between Vehicle and NPCs groups 21 and 60 days after Crush, p<0.01 **.
We are gratefull to Mrs Ajdari For technical support and Royan Institute for financial support.
Fig 2: work time line and animal model confirmation. A. Time line of cell or medium injection, and tissue collection. B. Animal model confirmation, Light microscopy, Toluiden blue for Intact group (right), and Model group (Left) show axon number decreases in Model group.