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Promotion on the
differentiation of retinal Müller cells into retinal ganglion cells by Brn-3b
Zhen-Kai Wu, Lan
Cao, Xue-Yong Zhang, Wei-Tao Song, Xiao-Bo Xia
Department of Ophthalmology,
Xiangya Hospital, Central South University, Changsha 410008, Hunan Province,
China
Correspondence to: Wei-Tao Song; Xiao-Bo Xia. Department of
Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China.
wtsong1980@126.com; xbxia21@163.com
Received:
2015-04-15 Accepted: 2015-11-24
Abstract
AIM: To investigate
the role of Brn-3b in differentiation process of stem cells derived from
retinal Müller cells into the ganglion cell.
METHODS: The passage
culture method of Müller cells from retina of newborn Sprague Dawley rats was
carried out by repeated incomplete pancreatic enzyme digestion method. The
cells were detected by fluorescence-activated cell sorter (FACS),
immunohistochemistry technology and reverse transcription-polymerase chain
reaction (RT-PCR) to determine the purity. The third passage of cells was
induced in the serum-free dedifferentiation medium. The expression of the
specific markers Ki-67 and nestin of retinal stem cells was measured by RT-PCR
and Western blot. The cell proliferation of retinal stem cells was detected by
5-ethynyl-2'-deoxyuridine (Edu) staining. The cells were randomly divided into
5 groups as follows: group A: Brn-3bsiRNA group; group B: Brn-3b control siRNA
group; group C: pGC-Brn-3b-green fluorescent protein (GFP) group; group D: pGC-GFP
group; group E: control group (without any handling). The purified Müller cells
were cultured for 3-7d, then, the percentage of ganglion cells was counted by
immunofluorescence staining.
RESULTS: FACS
demonstrated the purity of retinal Müller cells was more 97.44%. A few
spherical cell spheres appeared. Immunofluorescence staining showed that stem
cells within the spheres were positive for retinal stem cell-specific markers
nestin (red fluorescence, 92.94%±6.48%) and Ki-67
(green fluorescence, 85.96%±6.04%). Meanwhile, RT-PCR analysis showed cell
spheres in the culture to have expressed a battery of transcripts
characteristic of stem cells such as nestin and Ki-67, which were absent in the
Müller cells. Western blot analysis further confirmed the expression of nestin
and Ki-67 in the cell spheres but not in the Müller cells. Edu staining showed
most of the nuclei within the cell spheres were stained red (82.80%±6.65%),
suggesting the new cell spheres had the capacity for effective proliferation.
The statistics result showed the difference between Brn-3bsiRNA group and
Brn-3b control siRNA group or the control group was significant (F=15, P＜0.05),
while the difference between Brn-3b control siRNA group or the control group
was not statistically significant (P＞0.05).
CONCLUSION: The repeated
incomplete pancreatic enzyme digestion method is an efficient and practical
method to purify retinal Müller cells. Retinal stem cells were successfully
cloned in the dedifferentiational medium. Retinal Müller cells are accessible
sources of retinal stem cells. Brn-3b is an important regulatory gene in stem
cells differentiated into retinal ganglion cell.
KEYWORDS: Müller cells;
retinal ganglion cells; Brn-3b; stem cells; differentiation
Citation: Wu ZK,
Cao L, Zhang XY, Song WT, Xia XB. Promotion on the differentiation of retinal
Müller cells into retinal ganglion cells by Brn-3b. Int J
Ophthalmol 2016;9(7):948-954
INTRODUCTION
Glaucoma
is a group of ocular disorders that can damage the eye’s optic nerve and result
in vision loss and blindness[1].
These lective and progressive death of retinal ganglion cells (RGCs) is a final
common feature of optic nerve damage of glaucoma[2]. Present numerous approaches have been developed to
provide protection to ganglion cells, but these approaches does not prevent the
visual loss caused by the death of RGCs[3-4].
In recent years, some preclinical studies started to find better treatments
from induced differentiation of RGCs. However, stem cells derived from retina
are limited in quantity, on the other hand, the use of other stem cells, such
as embryonic stem cells and neural stem cells, is greatly restricted due to
ethical issues, graft rejection and low efficiency of differentiation.
Therefore, if some kind of cells which are abundant in quantity and also
derived from the retinal cells can be found to be induces differentiation to
RGCs, it will afford more effective cure for the clinical gene-therapy of
glaucoma.
Recent
studies refers that retinal Müller cells were an accessible source of retinal
stem cells, which showed the same characteristics of RGCs in some particular
situations[5-6]. Müller
cells derived from retinal cells were abundant in seed cells without the
disadvantages of rejection reaction or causing ethical issue. The induction and
differentiation of retinal stem cells are largely regulated by the some
extracellular and intracellular factors. Recent studies[7]
have shown that the Brn-3b transcription factor, a pit-oct-unc (POU)
IV-class protein, is a member of the proteins targeted by the notch pathway during RGCs development. This
transcription factor plays a key role in RGC differentiation, survival, and
axon outgrowth. Previous study[8]
found that in the absence of Brn-3b, the affected ganglion cell precursors fail
to appear during development and that the modest reduction in cell number in
the inner and outer nuclear layers of the mature retina may follow as a
secondary effect of reduced ganglion cell number. In this research, by
purifying and cloning retinal Müller cells, inducing them to dedifferentiate
into RGCs, we studied the regulatory influences of Brn-3b in the
differentiation of retinal stem cells derived from Müller cells into RGCs.
MATERIALS AND METHODS
Experimental
Animals Newborn
Sprague Dawley (SD) rats of either gender. The experimental animals in this
study was in accordance with the Guidelines for Animal Experiments of Central
South University, Changsha, China. All animal experiments in this study were
conducted with the approval of the Animal Research Committee, Xiangya School of
Medicine, Central South University, Changsha, China (Permit No. SCXK
2006-0002).
Methods
Purification and culture of Müller
cells The eye balls
from a total of 10 newborn SD rats, were enucleated after sacrifice by soaking
in 75% ethyl alcohol. Their retinas were removed carefully. The culture method
of Müller cells was carried out by repeated incomplete pancreatic enzyme
digestion method. The first, second, third passage of retinal Müller cells was
detected respectively by fluorescence-activated cell sorter (FACS),
reverse-transcription polymerase chain reaction (RT-PCR), Western blot and
immunohistochemistry technology to determine the purity by testing the
glutamine synthetase (GS), which is a specific marker to identify Müller cells.
Induced dedifferentiation and
neurospheres generation of Müller cells
After
washing the third passage of retinal Müller cells isolated from the medium in
phosphate buffer solution for three times, we dissociated the cells with 0.25%
trypsin-EDTA in a 37℃ tank for 3min. The digested retina was suspended in
Dulbecco's modified Eagle’s media: nutrient mixture F-12 (DMEM/F12) (1:1)
(Grand Island Biological Company, GIBCO), 1×N2
supplement (GIBCO), 2×B27 supplement (GIBCO), 20 ng/mL epidermal growth factor
(EGF, Peprotech), 10 ng/mL basic fibroblast growth factor (bFGF, Peprotech), 2
mmol/L glutamine , 100 U/mL penicillin and 100 μg/mL streptomycin until the
adherent cells became round under microscopy. The cell suspensions were collected
in 15 mL centrifugal tube and the liquid was centrifuged for 3min at 800 r/min.
We discarded the liquid supernatant and add dedifferentiated medium to dilute
the cells at a density of 1×105 cells/cm2. The cells were
seeded into twelve-well culture plates, cultured at 37℃ in a 5% CO2
incubator. Half of the dedifferentiation media was changed every 3d. We
observed the generation state of these cells and detected the specific marker
of Müller cells in different time by FACS, RT-PCR, Western blot and 5-ethynyl-2'-deoxyuridine
(Edu) staining to determine the multiplication capacity.
Differentiation of retinal stem
cells The neurospheres
suspension cells were collected and centrifuged them for 3min at 500 r/min. We
discarded the liquid supernatant and add appropriate amount of Accutase to
dissociate them to single cell. These cells were centrifuged again and
resuspended with DMEM/F12 differentiation medium containing brain derived
neurophic factor (BDNF) (1 ng/mL), retinoic acid (RA) (1 μmol/L) and 1% fetal
bovine serum (FBS). The cells were randomly divided into 5 groups as follows:
group A: Brn-3bsiRNA group (sc-38767, American Abcam Company); group B: Brn-3b
control siRNA group (sc-37007, American Abcam Company); group C:
pGC-Brn-3b-green fluorescent protein (GFP) group (Changsha Aijiabio technology
Co. Limited); group D: pGC-GFP group (Changsha Aijiabio technology Co.
Limited); group E: Control group (without any handling). The stem cells were
intervened referred to laboratory manual and cultured in a 37℃ incubator.
RT-PCR, Western blot and immunohistochemistry technology were performed to
detect the percentage of ganglion cells in total differentiated cells after 7d.
Cell Counting and Statistical Analysis Ten
non-overlapping visual fields in each group were randomly selected under a
fluorescence microscope (×10) to count the number of Müller cells. All data
were expressed as the mean±SD. Statistical analysis was performed with one-way
ANOVA and Student’s t-test in SPSS
13.0. The difference was statistically significant if P<0.05.
RESULTS
Purity of Müller Cells FACS
demonstrated that the purity of the first passage retinal Müller cells was
70.89% (Figure 1A), the second passage retinal Müller cells was 90.17% (Figure
1B), the third passage retinal Müller cells was more than 97.44% (Figure 1C).
Taking
the first, second, third passage of Müller cells for immunofluorescence
staining, we found that, as the cells proliferated, the amount of unwanted
cells decreased, and the Müller cells tended to be in the same size and shape.
Their cytoplasm became more abundant and the non-specific fluorescence
decreased. The density of cultured cells increased and they were equally
distributed (Figure 1D-1F). Ten non-overlapping visual fields in each group
were randomly selected under a microscope (×10) to count the
immunoreactivity for GS. The purity of the first, second, third passage of
Müller cells were 73.45%±2.47%, 94.35%±1.26%, 98.50%±1.08% respectively (Figure
1G).
To further
determine the purity of cultured cells, we carried out RT-PCR and Western blot
analysis to detect the specific markers GS, taking the retinal tissue as a
control. The results indicated that large amount of GS are expressed in both
retinal tissues and purified Müller cells, while the GS amount of Müller cells
were at a higher level; and as the cells proliferated, the GS amount rapidly
increased (Figure 1H).
Figure 1 Purity of Müller Cells FACS
demonstrated the purity of retinal Müller cells. A: The first passage was
70.89%; B: The second passage was 90.17%; C: The third passage was more than
97.44%; dual staining of primary
culture of retinal Müller cells at different passage for
4,6-diamino-2-phenylindole (DAPI) and GS (×100); D: The first passage; E: The
second passage; F: The third passage; the nuclei were stained with DAPI; G: As
the cells proliferated, the density of cultured cells increased and they were
equally distributed; the purity of the first, second, third passage of Müller
cells were 73.45%±2.47%, 94.35%±1.26%, 98.50%±1.08% respectively; H: RT-PCR and Western blot analysis
indicated that large amount of GS are expressed in both retinal tissues and
purified Müller cells,while the GS amount of Müller cells were at a higher
level; and as the cells proliferated, the GS amount rapidly increased.
Characteristics of Müller Cells During
Dedifferentiation and Proliferation
Characteristics of Müller cells during
induced dedifferentiation The
purified Müller cells were cultured in stem-cell-conditioned medium for 3d. The
cells became round and big, abundant of cytoplasm, with clear outlines and high
refraction. The proliferation was clonal, and dozens of cells formed cell
spheres suspending at the base of the medium, living in suspension (Figure 2A).
We changed half of the dedifferentiation media carefully every 2d to avoid
removing the cell spheres. At the 5th day of culture, the cell
spheres increased in both number and size, and cells exhibited clear
boundaries. At the edge of some cell spheres, we can see some short burrs-like
protuberances with high refraction (Figure 2B). At the 7th day of
culture, the cell spheres showed no significant increase in number and size
(Figure 2C).
Detection of specific markers of cell
spheres and the capability of proliferation Taking the
cultured cell spheres of the 7th day for immunofluorescence
staining, we detected the specific markers, nestin and Ki-67. The result showed
that the cell spheres expressed a battery of nestin (red fluorescence,
92.94%±6.48%) and Ki-67 (green fluorescence, 85.96%±6.04%), which are specific
markers of neural stem cells (Figure 2D). Meanwhile, Edu staining showed that
most of the nuclei within the cell spheres were positive and stained red
(82.80%±6.65%), suggesting that the cell spheres have the capacity for
effective proliferation (Figure 2E).
Expression of specific markers and mRNA
within cell spheres Taking
the cultured cell spheres of the 7th day for RT-PCR and Western blot
analysis, the high expression of nestin, Ki-67 and mRNA within cell spheres,
compared them with the expression within purified Müller cells (Figure 2F, 2G).
Figure 2 Cell spheres derived from
Müller cells A: At
the 3rd day
of culture, the cells became round and big, abundant of cytoplasm, with clear
outlines and high refraction. The proliferation was clonal, and dozens of cells
formed cell spheres suspending at the base of the medium, living in suspension;
B: At the 5th day of culture, the cell spheres increased in both
number and size, and cells exhibited clear boundaries. At the edge of some cell
spheres, we can see some short burrs-like protuberances with high refraction;
C: At the 7th day of culture, the cell spheres showed no significant
increase in number and size (bar=200 µm); D: The
results of immunofluorescence staining showed the cell spheres expressed a
battery of Nestin (red fluorescence, 92.94%±6.48%) and Ki-67 (green
fluorescence, 85.96%±6.04%); E: Edu staining showed that most of the nuclei
within the cell spheres were positive and stained red (82.80%±6.65%),
suggesting that the cell spheres have the capacity for effective proliferation
(bar=200 µm); F: High expression of nestin, Ki-67 protein within cell spheres
(1: purified Müller cells; 2: cell spheres); G: High expression of nestin,
Ki-67mRNA within cell spheres (1: purified Müller cells; 2: cell spheres).
Promotion on the
differentiation of retinal Müller cells into retinal ganglion cells by Brn-3b After
intervention with the 5 groups and cultured for 7d, immunocytochemical analysis
was performed to calculate the percentage of ganglion cells differentiated from
retinal stem cells derived from Müller cells in ten randomly selected
non-overlapping visual fields(the percentage of ganglion cells differentiated
from retinal stem cells=numbers of cells positive for Thy1.1 and Brn-3b/numbers
of cell nucleus positive for DAPI×100%). The results
showed that the percentage of ganglion cells differentiated from retinal stem
cells of group A: 2.79%±0.32%, group B: 13.64%±1.26%, group C: 33.63%±4.27%, group
D: 12.20%±1.32%, group E: 13.80%±1.04% (Figure 3). The statistics result showed
the difference among group B, D, E was not statistically significant (P＞0.05), suggesting that Brn-3b control siRNA and the
control had no influence on the differentiation of retinal stem cells derived
from Müller cells into RGCs; while the difference was significant between Group
A and B, C, D, E respectively (F=15, P=0.001), the
difference was significant between group C and A, B, D, E respectively (F=34, P=0.001), suggesting that Brn-3b acting as an important
regulatory factor, promoted the differentiation of retinal stem cells derived
from Müller cells into RGCs.
Figure 3 The percentage of ganglion
cells differentiated from retinal stem cells The percentage
of ganglion cells differentiated from retinal stem cells of Brn-3bsiRNA group:
2.79%±0.32%; control siRNA group: 13.64%±1.26%; pGC-Brn-3b-GFP group:
33.63%±4.27%; pGC-GFP group: 12.20%±1.32%; control group: 13.80%±1.04%. The
statistics result showed the difference between Brn-3bsiRNA group and Brn-3b
control siRNA group or the control group was significant (F=15, P＜0.05), while the
difference between Brn-3b control siRNA group or the control group was not
statistically significant (P＞0.05). The
difference was significant between group C and A, B, D, E respectively (F=34, P=0.001).
DISCUSSION
Retinal
cells include six neurons and one neurogliocyte: ganglion cells, cone cells,
horizontal cells, amacrine cells, rod cells, bipolar cells and Müller cells.
Müller cells are a special type of neurogliocyte. On the development of time,
the first three cells developed earlier, and the last one is Müller cells.
Müller cells and other six neurons share the same progenitor cell[9-10]. Müller cells are
radial glial cells which span the entire depth of the neural retina. Radiating
from the soma (in the inner nuclear layer) is an inwardly directed process that
terminates in an expanded end foot at the inner border of the retina, adjacent
to the vitreous humor. Also projecting from the soma is an outwardly directed
process that ends in the photoreceptor layer. Microvilli project from this
apical process into the subretinal space surrounding the photoreceptors[11-13]. In function, Müller cells have great influences in the following
aspects: 1) Müller cells induce the transference, differentiation of
retinal neurons and help to maintain their subsistence, promote the development
of nervous process, build the retinal neural network during embryonic period;
2) Müller cells nourish retinal neurons and remove their metabolite; 3) Müller
cells regulate the neuronal micro-environment including water, PH, K+ in the
extracellular fluid bathing central nervous system cells; 4) Müller cells play
an important role in removing neurotransmitters from extracellular space
following their release from synaptic terminals; 5) by controlling the
concentration of neuroactive substances in extracellular space, Müller cells
can significantly modulate neuronal activity. Recent studies refers that Müller
cells were an accessible source of retinal stem cells, which showed the same
characteristics of RGCs in some particular situations, there for Müller cells
have become the focus in the generation of retinal neurons.
Retinal
tissues contain many types of cell ingredients, we developed a modified
approach called repeated incomplete pancreatic enzyme digestion to purify
Müller cells. At present, the tissue culture and enzyme digestion methods are
the main ways of purifying Müller cells[14].
With the tissue culture method, retinal tissue is dissociated into small
aggregates and the cells attached to the walls, which have been taken from
tissues, are passaged. This method generally does not yield high-purity Müller
cells, or if it does, the culturing time required is too long. In the enzyme
digestion method, cells are passaged with pancreatic enzyme, which is able to
detach cells from walls. Then cells are suspended digestion and centrifuged,
cultured in medium. In the process of purifying retinal Müller cells from SD
rats, the other retinal cells are removed after repeated light pancreatic
enzyme digestion based on the characteristics of Müller cells that they can
attach to walls quickly and firmly. Using this method, we were able to obtain
plenty of high-purity Müller cells, which establish the foundation for further
study on the Müller cells. In our study, the purity of the third passage
retinal Müller cells was more than 95%, which showed that the repeated
incomplete pancreatic enzyme digestion method is an efficient and practical
method to purify retinal Müller cells.
GS is
a key enzyme that transfers glutamic acid into glutamine and is only expressed
in Müller cells[15]. Therefore,
we chose GS as the specific marker to identify Müller cells. Except GS, Müller
cells also express vimentin, clusterin, however, both of the proteins are also
expressed in other cells. Then we could also identify Müller cells by detecting
the relatively specific markers. The RT-PCR results showed that retina Müller
cells of SD rat expressed large amount of GS, vimentin, clusterin; in contrast,
specific markers corresponding to other types of cells of retinal tissue, such
as opsin (rodcells), mGluR6 (bipolar cells), syntaxin 1 (amacrine cells),
Brn-3b (RGCs), tyrosinase (pigmented ciliary epithelium cells) and CD31
(endothelial cells) were not detected in purified Müller cells. By counting the
immunoreactivity for GS, we detected the purity of the first, second, third
passage of Müller cells were 73.45%±2.47%, 94.35%±1.26%, and 98.50%±1.08%
respectively. These results demonstrate that our method of purifying Müller
cells from retinal tissues is effective.
Purified
Müller cells were cultured in a serum-free DMEM/F12 medium supplemented with
EGF, bFGF and other nerve growth factor including N2 and B27 to induce the
dedifferentiation of retinal Müller cells to neural-stem-cells-like cell
spheres. A battery of nestin (green fluorescence, 92.94%±6.48%) and Ki-67 (red
fluorescence, 85.96%±6.04%) of the cell spheres in immunofluorescence showed
that the cells within the neuro spheres highly expressed retinal stem
cell-specific markers nestin and Ki-67; Western blot and RT-PCR analysis showed
that cell spheres in the culture expressed a battery of transcripts
characteristic of stem cells such as nestin and Ki-67; Edu staining showed that
nuclei within the cell spheres were stained strongly positively (82.80%±6.65%),
suggesting that Müller cells can differentiate to retinal stem cells and have
the capacity for effective proliferation.
EGF
and bFGF are important mitogens in cell proliferation, regulating the activity
of cell proliferation. Study in vitro
showed that the amount of Edu-marked cells increased which cultured in medium
with EGF. Retinal precursor cell of embryonic rat cultured with EGF could
differentiate to retinal neural cells and gliocyte, indicating that EGF could
regulate the proliferation of neural precursor cells[16]. bFGF is a mitogen of neural precursor cells in
rodent. These neural precursor cells are from embryonic retina, cortex,
hippocampus, corpusstriatum, midbrain, spinalcord and the subventricular zone
of adult brain[17]. bFGF,
EGF and other cytokines together regulate the proliferation neural stem cells.
Our results proved that purified Müller cells cultured in DMEM\F12 medium and
supplemented with EGF, bFGF and other nerve growth factor including N2 and B27
can be effectively induced dedifferentiation to retinal stem cells, which
establishes the foundation for further study on the differentiation of retinal
stem cell.
Retinal
stem cells differentiate naturally to any kind of retinal cells in medium for
differentiation, which is regulated by several genes. Among these regulatory
factors, Brn-3bcan synergize to promote the growth and differentiation of rat
RGCs during the embryo period, promoting the directed differentiation of
embryonic stem cell into RGCs[18-20].
To investigate whether Brn-3b has the same regulatory effects on retinal stem cells
besides embryonic stem cells, we used Brn-3bsiRNA to knockout Brn-3b gene in
Müller cells-derived stem cells. Brn-3bsiRNA targets Brn-3bmRNA in a RGC thus
potentially killing the cells expressing it[21].
The results showed that the differentiation rate of Müller cells-derived stem
cells into RGCs decreased, confirming the crucial role of Brn-3b gene in
promoting the directed differentiation of stem cells into ganglion cells. Our
study demonstrates that we can use Brn-3b gene to induce the directed differentiation
of retinal stem cells derived from Müller cells into RGCs efficiently, which
offers a new method for gene replacement therapy and optic nerve regeneration
in glaucoma. At the same time, the differentiation mechanism and regulatory
network of stem cells are complicated, which is worth further investigating.
ACKNOWLEDGEMENTS
Wu ZK:
Collection and assembly of data, data analysis and interpretation, manuscript
writing; Cao L and Zhang XY: Collection and assembly of data; Song WT and Xia
XB: Corresponding author, conception and design, financial support, final
approval of manuscript. All authors read and approved the final manuscript.
We are grateful
to Dr. Ying-Qun Wang for his valuable advice and guidance; Dr. Si-Qi Xiong for
their helpful suggestion; Prof. Hong Shen, Dr. Jing-Yu He, and Dr. Sai Zhang
for technical assistance.
Foundation:
Supported
by National Natural Science Foundation of China (No.81400400).
Conflicts of Interest: Wu ZK, None; Cao L, None; Zhang XY, None; Song WT, None; Xia XB, None.
REFERENCES
1 Quigley HA, Broman AT. The number of people with glaucoma
worldwide in 2010 and 2020. Br J
Ophthalmol 2006;90(3):262-267. [CrossRef] [PubMed] [PMC free article]
2 He M, Foster PJ, Ge J, Huang W, Zheng Y, Friedman DS, Lee PS,
Khaw PT. Prevalence and clinical characteristics of glaucoma in adult Chinese:
apopulation-based study in Liwan District, Guangzhou. Invest Ophthalmol Vis Sci 2006;47(7):2782-2788. [CrossRef] [PubMed]
3 Locker M, EI Yakoubi W, Mazurier N, Dullin JP, Perron M. A
decade of mammalian retinal stem cell research. Arch ItalBiol 2010;148(2):59-72. [PubMed]
4 Bringmann A, Pannicke T, Grosche J, Francke M, Wiedemann P,
Skatchkov SN, Osborne NN, Reichenbach A. Müller cells in the healthy and
diseased retina. Prog Retin Eye Res 2006;25(4):397-424.
[CrossRef] [PubMed]
5 Nickerson PE, Da Silva N, Myers T, Stevens K, Clarke DB. Neural
progenitor potential in cultured Müller glial: effects of passaging and
exogenous growth factor exposure. Brain
Res 2008;1230:1-12. [CrossRef] [PubMed]
6 Bernardos RL, Barthel LK, Meyers JR, Raymond PA. Late-stage
neuronal progenitors in the retina are radial Müller glial that function as
retinal stem cells. J Neurosci 2007;27(26):7028-7040.
[CrossRef] [PubMed]
7 Calissano M, Diss JK, Latchman DS. Post-transcriptional
regulation of the Brn-3b transcription factor in differentiating neuroblastoma
cells. FEBS Lett
2007;581(13):2490-2496. [CrossRef] [PubMed]
8 Gan L, Xiang M, Zhou L, Wagner DS, Klein WH, Nathans J. POU
domain factor Brn-3b is required for the development of a large set of retinal
ganglion cells. Proc Natl Acad Sci U S A 1996;93(9):3920-3925.
[CrossRef]
9 Giannelli SG, Demontis GC, Pertile G, Rama P, Broccoli V. Adult
human Müller glia cells are a highly efficient source of rod photoreceptors. Stem Cells 2011;29(2):244-356. [CrossRef] [PubMed]
10 Cho MS, Kim SJ, Ku SY, Park JH, Lee H, Yoo DH, Park UC, Song
SA, Choi YM, Yu HG. Generation of retinal pigment epithelial cells from human
embryonic stem cell-derived spherical neural masses. Stem Cell Res 2012;9(2):101-109. [CrossRef] [PubMed]
11 Sapkota D, Wu F, Mu X. Focus on molecules: Math5 and retinal
ganglion cells. Exp Eye Res 2011;93(6):796-797. [CrossRef] [PubMed] [PMC free article]
12 Fimbel SM, Montgomery JE, Burket CT, Hyde DR. Regeneration of
inner retinal neurons after intravitreal injection of ouabain in zebrafish. J Neurosci 2007;27(7):1712-1724. [CrossRef] [PubMed]
13 Song WT, Zhang XY, Xiong SQ, Wen D, Jiang J, Xia XB. Comparison
of two methods used to culture and purify rat retinal Müller cells. Int J Ophthalmol 2013;6(6):778-784. [PMC free article]
[PubMed]
14 Song WT, Zhang XY, Xiao XB. Atoh7 promotes the differentiation
of retinal stem cells derived from Müller cells into retinal ganglion cells by
inhibiting Notch signaling. Stem Cell Res
Ther 2013;4(4):94. [CrossRef]
[PubMed] [PMC free article]
15 Yurco P, Cameron DA. Responses of Müller glia to retinal injury
in adult zebrafish. Vision Res
2005;45(8):991-1002. [CrossRef]
[PubMed]
16 Parrilla M, Lillo C, Herrero-Turrión MJ, Arévalo R, Aijón J,
Lara JM, Velasco A. Characterization of Pax2 expression in the goldfish optic
nerve head during retina regeneration. PLoS
One 2012;7(2):e32348. [CrossRef] [PubMed] [PMC free article]
17 Ara J, Fekete S, Zhu A, Frank M. Characterization of neural
stem/progenitor cells expressing VEGF and its receptors in the subventricular
zone of newborn piglet brain. Neurochem
Res 2010;35(9):1455-1470. [CrossRef] [PubMed]
18 Karl MO, Reh TA. Regenerative medicine for retinal diseases:
activating endogenous repair mechanisms. Trends
Mol Med 2010;16(4):193-202. [CrossRef] [PubMed] [PMC free article]
19 Ho T, Vessey KA, Fletcher EL. Immunolocalization of the P2X4
receptor on neurons and glia in the mammalian retina. Neuroscience 2014;277:55-71. [CrossRef] [PubMed]
20 Das AV, Mallya KB, Zhao X, Ahmad F, Bhattacharya S, Thoreson
WB, Hegde GV, Ahmad I. Neural stem cell properties of Müller glia in the
mammalian retina: regulation by Notch and Wnt signaling. Dev Biol 2006;299(1):283-302. [CrossRef] [PubMed]
21 Calissano M, Latchman DS. Cell-specific regulation of the
pro-survival Brn-3b transcription factor by micro RNAs. Mol Cell Neurosci 2010;45(4):317-323. [CrossRef] [PubMed]
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