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Journal of Cell Science
© 2015. Published by The Company of Biologists Ltd.
C-kit+ Cells Isolated from Human Fetal Retinas Represent a New Population of Retinal
Progenitor Cells
Pengyi Zhoua, Guang-Hua Penga,b, Haiwei Xuc,d, Zheng Qin Yinc,d
a.Department of Ophthalmology, The First Affiliated Hosptial of Zhengzhou University, Zhengzhou, He’nan, China
b. Department of Ophthalmology, General Hospital of Chinese People’s Liberation Army,Beijing, China
c.Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing, China;
d.Key Lab of Ophthalmology of Chinese People’s Liberation Army, Chongqing, China
Author Contributions:
Peng Yi Zhou: Conception and design, Data collection and analysis, Manuscript writing
Guang-Hua Peng: Conception and design, Data analysis and interpretation, Provision of study material,
Manuscript writing.
Hai Wei Xu: Design, Data analysis and interpretation
Zheng Qin Yin: Conception and design, Provision of study material
Corresponding Authors：
Prof. Guang-Hua Peng, The First Affiliated Hosptial of Zhengzhou University, Zhengzhou, 450052; General
Hospital of Chinese People’s Liberation Army,Beijing, 100853 (China), Phone: +86-01066937284. Fax:
86-01068182168; E-Mail ghp@zzu.edu.cn; Competing interests: The authors declare that they have no competing
interests.;
Prof. Zheng Qin Yin，Southwest Eye Hospital, Southwest Hospital, Third Military Medical University;Key Lab of
Ophthalmology
of
Chinese
People’s
Liberation
Army,
Chongqing,
China;TEL.+86-2368754803,E-Mail
yzhengqin@163.com
Keywords: Fetal·Retinal progenitor cells·C-kit·Transplantation·Retinal degeneration
JCS Advance Online Article. Posted on 27 April 2015
Abstract
Definitive surface markers for retinal progenitor cells (RPCs) are still lacking. Therefore, we
sorted C-kit+ and stage-specific embryonic antigen-4 (SSEA-4-) retinal cells for
further biological characterization. RPCs were isolated from human fetal retinas (gestational
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sorting, and their proliferation and differentiation capabilities were evaluated by
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age of 12 to 14 weeks). C-kit+/SSEA4- RPCs were sorted by fluorescence-activated cell
proliferation potential of these cells. Moreover, C-kit+/SSEA4- cells differentiated into retinal
immunocytochemistry
and
flow
cytometry.
The
effectiveness
and
safety
were
assessed following injection of C-kit+/SSEA-4- cells into the subretina of Royal College of
Surgeons (RCS) rats.
C-kit+ cells were found in the inner part of the fetal retina. Sorted
C-kit+/SSEA4- cells expressed retinal stem cell markers. Our results clearly demonstrated the
cells that expressed markers of photoreceptor cells, ganglion cells, and glial cells. These cells
survived for at least 3 months after transplantation into the host sub-retinal space. Teratomas
were not observed in the C-kit+/SSEA4- cell group. Thus, C-kit can be used as a surface
marker for RPCs, and C-kit+/SSEA4- RPCs exhibited the ability to self-renew and
differentiate into retinal cells.
Introduction
Photoreceptor degeneration occurs as a result of disorders affecting either the
photoreceptors themselves or the associated retinal pigment epithelium (RPE) cells. This
disease is a common cause of blindness and severely affects a person’s quality of life(Pinilla
et al., 2004b). Photoreceptor degeneration is difficult to treat because the pathological process
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photoreceptor cells cannot regenerate or self-repair. Although RPE cells have the capacity to
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involves the apoptosis of photoreceptors or retinal pigment epithelium cells, and
degeneration is the key to treatment. Recently, stem cell-based cell therapy has become a hot
proliferate in vivo and in vitro(Chiba, 2014; Stanzel et al., 2014), it is difficult for damaged
RPE cells to repair themselves(Chiba, 2014). Currently, there are several treatment methods
available, including gene therapy, transplantation therapy, drug therapy, and artificial vision
prostheses. Rescuing or regenerating photoreceptor cells or RPE in patients with retinal
topic. Schwartz reported the safety and tolerability of human embryonic stem
cells(hESC)-derived RPE cells for the treatment of dry age-related macular degeneration
(AMD) and Stargardt’s disease; this report was the first description of the transplantation of
hESC-derived cells into human patients (Schwartz et al., 2012) (Schwartz et al., 2015).
Moreover, the authors observed no adverse proliferation or systemic safety issues related to
the transplanted cells, and the best-corrected visual acuity was improved in some patients
(Schwartz et al., 2012) (Schwartz et al., 2015). Thus, this human ESC-based cell therapy in
the blindness causing diseases is of highly significant for it provide the bright future for cell
therapy in anydisease(Sowden, 2014).
Tissue-specific retinal progenitor cells (RPCs) are an ideal source of differentiated cells
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with low tumour risk(Kajstura et al., 2011). Currently, most RPCs used for transplantation are
derived from ESCs or isolated from fetal tissues, but ESCs are difficult to differentiate in vitro,
are not easily purified, and may contain a variety of cell types or cells at different
development stages. The continuous development of flow cytometry to assess cell surface
antigens has provided biomarkers that can be used to obtain highly purified tissue-specific
RPCs. To date, several cell markers have proven to be suitable for the specific identification,
isolation, and enrichment of RPCs(Carter et al., 2009; Koso et al., 2009); these markers are
invaluable for RPC research.
Stem cell factor receptor C-kit (CD117), a progenitor cell marker, is a recognized antigen
located on the cell surface that plays an important role in the survival, proliferation, and
anti-apoptosis of haematopoietic stem cells (HSCs) and lung stem cells(Ellison et al., 2013).
Koso(Koso et al., 2007) identified C-kit as a RPC marker in the mouse retina, and
demonstrated a dramatic change in the expression profiles of the cell surface antigens C-kit
and stage-specific embryonic antigen (SSEA) on RPCs during development. Hasegawa
demonstrated that the human embryonic retina has a pool of C-kit+ cells; however, the authors
did not further culture and characterize them(Hasegawa et al., 2008). Stage-specific
embryonic antigen-4 (SSEA-4), a human embryonic stem cell-associated antigen(Wright and
Andrews, 2009), has previously been used as a marker to distinguish primitive embryonic
stem cells(Kawanabe et al., 2012). A sub-population of c-kit+ cells expressing SSEA-1
showed higher proliferative potential than c-kit+/SSEA1- cells(Koso et al., 2007). However, in
contrast to mouse ESCs, human ESCs lack SSEA1 and express SSEA4(Wright and Andrews,
2009). Therefore, to reduce the risk of tumourigenicity we used SSEA-4 as a surface marker
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to exclude cells with high proliferative potential isolated from the retina of human fetuses
using fluorescence-activated cell sorting.
We hypothesized that c-kit+ cells isolated from fetal retina tissues represent a population
of stem cells. The RPCs were evaluated for cell characteristics including self-renewal capacity,
clonogenicity, the ability to differentiate into three types of retinal cells in vitro, and the ability
to differentiate into photoreceptors in vivo. Additionally, we injected the cells into the
subretinal space of Royal College of Surgeon (RCS) rats, an animal model in which vision
deteriorates due to RPE dysfunction; these rats serve as a model for a recessive mutation in a
receptor tyrosine kinase gene (Mertk) that results in the progressive death of the
photoreceptors(Pinilla et al., 2004b). Then, we observed the survival, migration, and
differentiation of the cells, and determined their role in photoreceptor rescue and its impact on
visual function. Finally, we assessed the safety of transplantation of C-kit+/SSEA-4- retinal
progenitor cells into severe combined immune deficiency (SCID) mice. In our study, we
found that the transplantation of C-kit+/SSEA4- cells derived from human fetal retina tissues
could protect the neural retina and preserve the retinal outer nuclear layer in RCS rats.
Therefore, this study describes a method that may be used to obtain tissue-specific RPCs that
could be used to delay the photoreceptor degeneartion and preserve the retinal outer nuclear
layer in an animal model of retinal degeneration.
Results
Distribution of C-kit+ cells in human fetal eyes
The fetal neural retina at 12-13 weeks mainly consists of two layers: the outer neuroblastic
layer (ONbL) and inner neuroblastic layer (INbL). The cells in the outer neuroblastic layer
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(ONbL) are densely packed, while the cells in the INbL are more loosely packed. The
ganglion cell layer can be seen around the optic disc, but not the peripheral retinal.
C-kit+ cells were not only distributed in the retina, but also in the cornea and choroid of the
eye from a 13-week-old human fetus. C-kit+ cells were localized in the inner retina from the
optic nerve to the ora serrata(Fig. S2), although more cells were located in the peripheral
portion than at the posterior pole of the retina; C-kit+ cells were also scattered in the
corneoscleral limbus of the cornea(Fig. S3) and choroid (Fig. 1A-C).
C-kit+/SSEA-4- cell isolation and culture
We found C-kit+ cells in the retinas of eyes from human fetuses. C-kit+/SSEA4- cells were
sorted directly from the fetal retina; however, these cells were difficult to culture. Therefore,
we first cultured retinal cells for 2-3 passages. Then, we collected retinal cells (a minimum of
5–10×106 cells) and sorted C-kit+/SSEA4- cells by FACS. These cells were collected and
plated in 24-well plates (5×103/cm2) (Fig. 1G). The morphology within the adherent
population was spindle-shaped or displayed various other shapes (Fig. 1H). In contrast,
spheres were formed when the cells were grown in serum-free proliferation medium (Fig. 1J).
The C-kit epitope remained detectable by immunofluorescence and FACS after passaging (Fig.
1I,K).
Characteristics of C-kit+/SSEA-4- cells
Cells stained for RPC markers including Pax6, Sox2, Rax, and Nestin were analysed by flow
cytometry and immunofluorescence. C-kit+/SSEA4- cells were found to express Pax6 (91.6±
2.7%), Sox2 (95.2±2.0%), Rax (94.1±2.5%), and nestin (98.9±2.8%) (Fig. 3). In contrast,
they did not express the embryonic stem cell/tumour cell markers SSEA4 and the multi drug
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resistance protein (MDR) (Fig. 2C, F). Because C-kit+ has also been described as a stem cell
marker in other organs (i.e., mesenchymal stem cells (MSCs) and HSCs), we stained the
sorted cells with MSC and HSC markers. The cells were negative for both the HSC markers
(CD11b and CD45) (Fig. 2B, H) and MSC markers (CD29 and CD140b) (Fig. 2E, I).
Characterization of proliferation
The cells exhibited the ability to proliferate and grow in a monolayer on plastic plates in
proliferation medium (Fig. 1H) and form spheres in serum-free proliferation medium (Fig. 1J).
Proliferating cells were identified by the proliferation marker ki67; the rate of ki67-positive
expression was determined to be 82.0±3.1% by FACS, which is consistent with the
immunofluorescence staining results (Fig. 4A, B). Cells were seeded into 24 well-plates at a
density of 10,000 cells/well, and the cell numbers were counted at 1,3,5,7, and 9 days after
seeding (n = 3). Cell proliferation reached a maximum and then plateaued after the cells were
plated; by 7 days, the cell number had increased by more than 20-fold (Fig. 4D). Additionally,
we examined the cell cycle distribution of the C-kit cells, and found that 41.13 ± 2.99% cells
were in the G2 and S phases (Fig. 4C).
C-kit+ cells were cultured under two different conditions: adherent conditions with
proliferation medium and non-adherent conditions with serum-free medium. When a single
C-kit+ cell (sorted using flow cytometry) was seeded into a 96-well plate, clones formed
approximately 20 days later (Fig. 1H). The clones were digested, 500 cells were seeded into
10 cm dishes, and more colonies were formed. When these cells were cultured in serum-free
medium, neurospheres formed after 20–30 days (Fig. 1J). The spheres were defined as
free-floating, with a diameter > 40 μm. The percentage of colony formation analysed after 30
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days was 0.06±0.01%，but the efficiency of colony formation was less than that observed
under adherent conditions（3.33%±1.53%）(P <0.01). This finding is consistent with the
results of a previous report(Rangel et al., 2013).
Characterization of differentiation
Multipotency is another property of stem cells. Therefore, we used two differentiation media:
photoreceptor differentiation medium and glia differentiation medium. The in vitro
differentiation was assessed by culturing C-kit+ cells in differentiation medium for 3 weeks
using the two types of differentiation media. Few differentiated cells expressed rhodopsin in
the differentiation medium. In contrast, many cells differentiated into glial cells (66.7±5.8%).
Because previous studies have shown that retinoic acid can promote the differentiation of
photoreceptor cells in vitro, we changed the medium for photoreceptor differentiation as
previously described(Nakano et al., 2012; Li et al., 2013).
Photoreceptor differentiation medium containing retinoic acid was used to culture
C-kit+/SSEA4- cells for 3 weeks. Then, we stained for photoreceptor cell markers (Otx2, Crx,
rhodopsin, and recoverin), a ganglion cell marker (Thy1), and a glial cell marker (GFAP) (Fig.
5G-L). Otx2 (49.9±4.1%), Crx (59.9±4.0%), Recoverin (68.1±5.1%), and Rhodopsin (4.0
±0.3%) were expressed in the differentiated cells, and 29.1±5.4% of the cells expressed
Thy1 (Fig. 5A-F). In contrast, the cells cultured in the initial differentiation medium only
expressed GFAP and Thy1.
Differentiation of grafted cells
Our immunostaining results showed that some cells migrated into the inner retina 8 and 12
weeks after transplantion, and a small amount of cells expressed Recoverin (Fig. 6H)(Fig. S1).
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However, the majority of the cells were located in the subretinal space. The percentage of
transplanted cells that expressed photoreceptor cell markers at 4w, 8w, and 12w were
1.01±0.11%，2.36±0.25%，5.22±0.14%, respectively.
Outer Nuclear Layer (ONL) Thickness
To verify the protective effect of transplanted cells on retinal degeneration, we measured
retinal ONL thickness.The ONL of the cell grafted retina was significantly thicker compared
to the control and untreated groups at 4 weeks (28.43±1.95μm vs. 7.67±1.08μm and
8.50±1.47μm, n=3, P <0.01), 8 weeks (23.27±0.85μm vs. 6.61±0.65μm, 6.83±1.08μm, n=3, P
<0.01), and 12 weeks (19.43±0.84μm vs. 4.17±0.75μm, 4.80±1.08μm, n=3, P<0.01). There
was no significant difference in ONL thickness between the control and untreated groups (Fig.
6M).
Electroretinogram (ERG) measurement
ERG was performed and b-waves were measured at 4 weeks, 8 weeks, and 12 weeks.
Significantly high amplitude b-waves were detected in the transplanted group at 4 and 8
weeks compared to the control and untreated groups (P <0.01). Recordings at 12 weeks were
not significantly different between the sham-surgery and untreated groups (P >0.05) (Fig.
6N-Q).
Teratoma assay
The safety of C-kit+/SSEA4- cells was tested by subcutaneous injection into the groin of six
SCID mice; hESCs were injected into another six SCID mice as a positive control. After 8
weeks, no gross inflammatory reaction was observed in any of the animals, and teratomas
were not observed in the C-kit+/SSEA4- cell group. In contrast, teratomas were seen in the
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hESC group at 8 weeks post-injection (Fig. 7).
Discussion
In 2011, the FDA approved phaseI/II clinical trials of cell transplantation therapy for
AMD and Stargardt’s disease, which led to immense advances in transplantation treatment of
retinal degeneration(Schwartz et al., 2012). RPC transplantation for the treatment of retinal
degenerative diseases has become the most promising therapeutic strategy. C-kit+ RPCs are
derived from fetal retinas and have advantages including the potential to differentiate into
retinal cells and low tumour risk. These factors make C-kit a good marker for the selection of
candidate cells for transplantation to treat retinal degeneration.
Our results showed that cells expressing the C-kit epitope on their cell surface were
distributed in the eye. C-kit+/SSEA4- cells possessed characteristics of self-renewal and the
ability to differentiate into three types of retinal cells. Due to the development of flow
cytometry technology and the discovery of new cell surface antigens, researchers have been
able to obtain higher purity stem cells by FACS.
C-kit defines a regionally and temporally
restricted immature subset of RPCs whose expression starts centrally and progresses
centrifugally(Koso et al., 2007).
At 9-10 fetal weeks, the retina was divided into outer neuroblastic layer (ONbL) and
inner neuroblastic layer (INbL). At 11-13 fetal weeks, the ganglion cell layer(GCL) was
several cell layers thick. A row of cones could be identified at the outer border of the outer
neuroblastic layer(ONbL) adjacent to the RPE, and a thin IPL separated the OnbL from the
differentiating GCL. The nerve fiber layer (NFL) was obvious on the inner border of the GCL
close to the optic disc. Recoverin and cones marker s-opsin expressed at 11-12 weeks, and
rods markers expressed at 15-16 weeks(O'Brien et al., 2003; Hendrickson et al., 2008). In our
study, fetal neural retina at 12-13 weeks mainly consists of two layers: the outer neuroblastic
layer (ONbL) and inner neuroblastic layer (INbL), which consist with fetal retinal
development report(O'Brien et al., 2003; Hendrickson et al., 2008). we found that C-kit+ cells
not only exist in the retina of the fetal eye, but are also located in the cornea and choroid; this
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cells were located in the inner layer of the retina; this finding is in agreement with the
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finding was not reported by Hasegawa(Hasegawa et al., 2008). Our results showed that C-kit
It is important to obtain pure RPCs and to exclude other types of embryonic stem cells
demonstration that the C-kit ligand SCF is also expressed in the inner retina(Hasegawa et al.,
2008). We successfully isolated and cultured C-kit+ cells derived from the human fetal retina
and identified their self-renewal, proliferation, and differentiation characteristics. We also
successfully used surface markers to isolate tissue-specific RPCs that do not express SSEA-4.
after isolation of eye tissue from the human fetus. The sorting of both the c-kit+ and SSEA-4surface markers enabled us to obtain purified RPCs. Additionally, the sorted cells were stained
with stem cell markers (MDR) by flow cytometry to ensure that there was no contamination
with embryonic stem cells; a total of 99% of the double-marker sorted cells did not express
the MDR marker.
C-kit+ cells were previously reported to be able to survive under suspension or adherent
growth conditions(Koso et al., 2007; Kajstura et al., 2011; Rangel et al., 2013). The C-kit
cells isolated in this study could also survive under both conditions. A higher proliferation
rate was observed under adherent conditions (in media supplemented with FBS), which is
consistent with Rangel’s report. One possible reason for this finding is that the cell-cell
interactions and cell adhesion present in adherent conditions are important for ckit+ cell
growth(Rangel et al., 2013). Retinal progenitor cells grown under adherent condition have
also been reported to exhibit a greater proliferative potential than cells grown under
suspension conditions(Xia et al., 2012).
To ensure that the c-kit+/SSEA4- cells sorted by FACS were highly tissue-specific, these
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Sox2 using immunohistochemistry and flow cytometry. More than 90% of the cells expressed
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cells were further evaluated for the human RPC tissue-specific markers nestin, Rax, Pax6, and
represented a highly pure and tissue-specific RPC with a certain proliferative capacity.
the RPC markers Pax6, Sox2, Rax, and nestin, indicating that the cells were in an immature
retinal cell state(Schmitt et al., 2009). Addditionally, cell proliferation potential was assesed
using the ki67 marker. More than 80% of the C-kit+/SSEA4- cells expressed this marker,
demonstrating that the c-kit+/SSEA4- cells possessed high proliferation ability in vitro and
Furthermore, we found that C-kit+/SSEA4- cells could form colonies under adherent and
suspension conditions. This finding is consistent with the other properties of C-kit cells
reported by Kajstura and Rangel(Kajstura et al., 2011; Rangel et al., 2013).
In addition to proliferation capability, another characteristic of progenitor cells is
differentiation capacity. In differentiation medium, c-kit+/SSEA4- cells could be induced into
photoreceptor cells, ganglion cells, and glial cells that expressed the corresponding
cell-specific markers (Otx2, Crx, Recoverin, Rhodopsin, Thy1, and GFAP). However, in our
study the proportion of cells expressing rhodopsin (4.0±0.3%) was lower than the proportion
reported in Coles`s study (34.5±9.1%); furthermore, we found that more cells differentiated
into glial cells (66.7±5.8%) than was previously reported (19.7±10.6%)(Coles, 2004).
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The transplantation of stem cells into the subretina of rats or mice retinal degeneration
has been demonstrated to produce a neuroprotective effect(Tian et al., 2011; Tzameret et al.,
2014). In our study, we found that transplantation of C-kit+/SSEA4- cells derived from human
fetal retina tissue could protect neural retinas in RCS rats. The RCS rat is characterized by a
recessive mutation in the Mertk gene, which is a receptor tyrosine kinase gene. This mutation
precludes RPE cells from phagocytosing shed rod outer segments, leading to the progressive
death of photoreceptor cells(Pinilla et al., 2004a). Morphological changes are already evident
by postnatal day (P)18 in pigmented RCS rats and include, among others, the disruption of
outer segments(Davidorf et al., 1991). Changes in photoreceptor nuclei are detected at P22
(Cuenca et al., 2005), and the rod contribution to the mixed b-wave starts declining after
P21(Pinilla et al., 2004a). We transplanted cells into the subretina at postnatal day 21, which
is when photoreceptor degeneration begins(Luo et al., 2014). Our experiment showed that
grafted C-kit+/SSEA4- cells could improve ERG b-wave amplitude, which represents the
electrical function of the retina(Tian et al., 2011), in RCS rats for 2 months after
transplantation; this finding is consistent with the results of the study by Tian(Tian et al.,
2011).
Anai(Gonzalez-Cordero et al., 2013) noted that reliable electroretinographic responses
were only achieved in mice following the rescue of 150,000 functioning rods. Our study
showed that ONL thickness was maintained for at least 3 months after cell transplantation;
however, the thickness decreased over time. This decrease is likely due to the fact that there
were not enough functional photoreceptors in the third month after transplantation. The
grafted cells expressed the photoreceptor marker recoverin. However, the migrated human
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RPCs failed to express the ganglion cell marker Thy1. It was previously reported(Luo et al.,
2014) that stem cells grafted into the degenerative retina had difficulty differentiating into
ganglion and photoreceptor cells in vivo; this finding was consistent with the results from our
study.
Finally, the major concern regarding the use of progenitor cells for transplantation is
tumourigenesis. The results from the teratoma assay showed that the transplantation of human
RPCs was safe. There was no evidence of tumour formation 8 weeks after human RPC
transplantation into the subretinal space of RCS rats.
In summary, our study demonstrated that C-kit+/SSEA4- cells exist in human fetal eyes,
and that these cells possess the stem cell properties of self-renewal, colony formation, and
pluripotent differentiation. Although only samll proportion of the engrafted C-kit+/SSEA4cells in a rat model of retinal degeneration could differentiate to express the photoreceptor
marker, C-kit+/SSEA4- cells delayed photoreceptor death by apoptosis or rescued host
photoreceptor cells for at least 3 months. Therefore, C-kit+/SSEA4- may serve as good
markers for selecting potential candidate cells for transplantation to delay retinal
degeneration.
Materials and Methods
Cell isolation and culture
Eyes from human fetuses with a gestational age ranging from 12 to 14 weeks were obtained
from spontaneous abortions at the Southwest Hospital, Third Military Medical University
(Chongqing, China). The Ethics Committee of Southwest Hospital specifically approved this
study, and it is registered in the Chinese Clinical Trial Register (ChiCTR; Registration number:
ChiCTR-TNRC-08000193). The gestational age of each fetus was determined using the last
menstruation date and fetal foot length(Merz et al., 2000; Drey et al., 2005). Postmortem
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times less than 1 hour were used because they do not alter the ability of progenitor cells in
culture(Carter et al., 2007).
Cells were isolated from the neuroretinas of human fetal eyes as previously
described(Coles, 2004; Klassen et al., 2004; Aftab et al., 2009; Schmitt et al., 2009; Baranov
et al., 2013). Briefly, the eyes were rinsed in cold Hank’s buffered salt solution (HBSS)
(Hyclone, South Logan, Utah, USA). The neuroretina was dissociated into small pieces and
enzymatically digested with 1 ml of papain (12 units/ml; Worthington, Lakewood, new jersey,
USA). The digested retinal tissue supernatant was filtered through a 40μm filter (BD
Biosciences, Franklin Lakes, New Jersey, USA) to obtain single cells. The cells were
centrifuged and re-suspended in proliferation medium supplemented with fetal bovine serum
(FBS). The isolated cells were seeded into 6-well plates at a density of 5×105 cells/well. The
cells were cultured at 37°C in the presence of 5% CO2, and the medium was changed every 3
days.
The following antibodies were used: APC-conjugated anti-human C-kit antibody (Biolegend,
San Diego, California, USA), FITC-conjugated anti-human SSEA-4 antibody (BD
Biosciences), PE-conjugated anti-human CD29 antibody (Biolegend), FITC-conjugated
anti-human multidrug resistance protein (MDR) antibody (Biolegend), APC-H7-conjugated
anti-human CD45 antibody (Biolegend), FITC-conjugated anti-human CD11b antibody (BD),
and PE-conjugated anti-human CD140b antibody (Biolegend).
The proliferation medium included Dulbecco's Modified Eagle Medium:Nutrient Mixture
F-12 (DMEM/F-12) (Hyclone) and supplemented with 20 ng/ml fibroblast growth
factor-basic (bFGF, PeproTech, Rocky Hill, NewJersey, USA), 20 ng/ml epidermal growth
factor
(EGF,
PeproTech),
1×insulin,
transferrin,
and
selenium
(ITS,
GIBCO),
1×penicillin-streptomycin (P-S, GIBCO, Carlsbad, California,USA), and 10% FBS (GIBCO).
Immunocytochemistry
Immunocytochemistry was performed as previously described(Tian et al., 2011; Pearson et al.,
2012). Briefly, rats were euthanatized with an overdose of anesthesia, and the eyes were
enucleated and fixed in 4% paraformaldehyde (0.01 M, pH 7.4). For human fetuses, the eyes
were enucleated and fixed in 2% paraformaldehyde (0.01 M, pH 7.4)(Hendrickson et al.,
2008). The eye cups were immersed in a graded series of sucrose solutions overnight,
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embedded in an optimal cutting temperature compound, and sectioned on a cryostat (Leica
CM190, Wetzlar, Germany). Frozen tissues sections were cut as 10m thick transverse
sections.
Immunocytochemistry was performed using our previously described methods(Duan et al.,
2013). Briefly, the cells or slides were fixed and permeabilized, then blocked in 10% serum
for 30 min. The cells were incubated with primary antibody at 37°C for 2 hours, washed with
PBS,
incubated
with
secondary
antibody,
washed
with
PBS,
incubated
with
6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature, and washed with PBS.
The following primary antibodies were used: anti-C-kit antibody (1:100, R&D Systems,
Minneapolis, Minnesota, USA), anti-Pax6 (1:200, Abcam, Cambridge, Massachusetts, USA),
anti-Sox2 (Abcam), anti-Rax (1:100, Abcam), anti-Nestin (1:200, Sigma, ST.Louis, Missouri,
USA), anti-Ki67 (1:300, Sigma), anti-GFAP (1:300, Abcam), anti-Thy1(1:100, BD
Biosciences), and anti-Recoverin (1:10,000, Millipore). The following secondary antibodies
were used: Cy3-conjugated antibody (1:800, Beyotime, NanTong, JiangShu, China) and
FITC-conjugated antibody (1:200, Beyotime). Additionally, the cells were stained with DAPI
(1:10, Beyotime). The images were captured using an Olympus OP70 microscope (Olympus
Microscopy, Japan) or Leica TCS SP50 confocal microscope (Leica Microsystems, Wetzlar,
Germany).
Fluorescence-activated cell sorting
The cells were digested with HyQtase for 5 min, followed by the addition of 3 ml of wash
buffer (Biolegend) and centrifugation at 400×g for 5 min at 4°C. Then, the cells were
re-suspended in Stain Buffer (Biolegend), and 2 µl of Fc block (Biolegend) was added to each
tube. The cells were incubated for 15 min at 4°C, and then incubated with fluorochrome
antibody conjugates for 20–30 min on a shaker at 4°C. The cells were washed with wash
buffer and centrifuged at 200×g for 5 min at 4°C. The supernatant was removed, 300l of
PBS was added to the cells, and the suspension was transferred to a standard flow cytometry
tube for fluorescence-activated cell sorting (BD Biosciences, AriaⅡ, Franklin Lakes, NJ,
USA).
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Expansion of C-kit+ cells
Sorted cells were plated at a density of 3-5×103/cm2 and cultured in proliferation medium
DMEM/F12 supplemented with 20% FBS, 20 ng/ml bFGF, 20 ng/ml EGF, and 10 ng/ml EGF
for 3 days. Then, the medium was changed and the FBS concentration was decreased to 10%.
The medium was changed every 3 days, and the cells were passaged until they reached 80%
confluency (every 3–4 days) at a constant seeding density of 10,000 to 13,000 cells/cm2.
Cell differentiation
Analysis of cell differentiation was performed using previously described methods(Coles,
2004; Li et al., 2013). Briefly, 5×103 cells/well of three passages of RPCs were seeded into
24-well plates on glass coverslips pre-coated with 0.015 mg/ml poly-lysine (Sigma) in glia
differentiation medium. For photoreceptor and other retinal cell differentiation, 5×103 cells
were seeded into 24-well plates on coverslips pre-coated with poly-L-lysine in photoreceptor
differentiation medium; the differentiation medium was changed every 4 days.
The glia differentiation medium consisted of DMEM/F12 (Hyclone) supplemented with 10
ng/ml bFGF (PeproTech), 1× P-S (GIBCO), 1% FBS (GIBCO), and 2μg/ml heparin (Sigma).
The photoreceptor differentiation medium consisted of DMEM/F12 (Hyclone) supplemented
with 10 ng/ml bFGF (PeproTech), 1× penicillin-streptomycin (GIBCO), 500 nM retinoic acid
(Sigma), and 2% B27 (GIBCO).
Cell proliferation curve
The cell proliferation assay was performed as previously described. Briefly, three passages of
RPCs were seeded into 15 wells of a 24-well plate at a density of 10,000 cells/cm2. The cell
number was counted in 3 wells at 1, 3, 5, 7, and 9 days; the experiment was repeated 3 times.
The average number of cells was used to generate the cell proliferation curve.
Animal feeding
The animals were treated as described under a protocol approved by the Institutional Animal
Care and Use Committee of the Third Military Medical University in accordance with the
NIH guidelines for the care and use of laboratory animals and the ARVO Statement for the
Use of Animals in Ophthalmic and Vision Research. Mice and rats were fed and housed under
a 12 hour light/dark cycle. The drinking water of the rats contained cyclosporine A (210 mg/l)
from 1 day before transplantation until they were sacrificed(Lu et al., 2010).
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Cell transplantation
Royal College of Surgeons (RCS) rats (males and females, 2–3 weeks old) were used for cell
transplantation. Rats with congenital microphthalmia, dysplasia of extremities, or congenital
cataracts were excluded from the study.
The RCS rats were randomly divided into two groups: the cell grafted group (n=9), in which
the rats received a subretinal injection of 3l of a C-kit+/SSEA4- cell suspension (cell
concentration, 2×105cells/l) and the PBS group (n=9), in which the rats received a subretinal
injection of 3l of HBSS. In both groups, the right eyes (OD) received cell transplants (cell
grafted group) or HBSS (group), while the left eyes (OS) were untreated. The left eyes of the
cell grafted group served as the untreated group.
Transplantation methods were performed as previously described(Tian et al., 2011). All rats
were anesthetized with a single intra-peritoneal injection of 4% chloral hydrate (0.8 ml/100 g
body weight). The pupils were dilated with 1% tropicamide. A Hamilton syringe (29 gauge;
Hamilton, Reno, NV, USA) containing the DiI (Invitrogen, Grand Island, New York,
USA)-labeled cell suspension was injected into the subretinal space.
Electroretinogram
Electroretinogram (ERG) techniques used performed as previously described(Tian et al.,
2011). The animals were tested at 4 weeks, 8 weeks, and 12 weeks. All rats were dark-adapted
overnight. The anesthesia method used was described in the Cell Transplantation section. A
Flash-ERG recording electrode, consisting of a small silver ring, was positioned on the
corneal surface with a drop of methyl cellulose and used to record responses (Roland system,
Wiesbaden, Germany). Each ERG response represents the average of three flashes. For all
Flash-ERG recordings, the b-wave amplitude was measured from the a-wave trough baseline
to the peak of the b-wave, and b-wave latency was measured from the onset of the stimulus to
the b-wave peak. ERG b waves were generated with flashes of white light at intensities of –
0.3 cds-1m-2 and 3.0 cds-1m-2.
Teratoma assay
C-kit+/SSEA4- cells (1×107 cells/100l) were injected into the groin in six severe combined
immune deficiency (SCID) mice and the animals were observed for 8 weeks to detect possible
tumour formation; hESCs were injected into six SCID mice as a positive control. The animals
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were sacrificed and examined by a pathologist to identify microscopic pathological changes
and evidence of tumour formation. The hESC cell line H-1 (WA-01) was kindly provided by
Professor Yue Huang(Ma et al., 2014) (School of Basic Medicine, Peking Union Medical
College, Beijing, China).
Analysis of the thickness of the outer nuclear layer
Three areas of retinal ONL thickness were examined in the transplanted area (but not in the
area that contained the layers of transplanted cells) in the treated and untreated groups and the
sham-surgery group. The thickness of the ONL was evaluated in DAPI-stained sections in
three areas along the grafted half of the retina.The ONL thickness was measured using
Image-Pro Express software.
Cell counts and analysis
The number of DiI and Recoverin double-positive cells in each image was counted at 3
locations in three areas along the grafted half of the retina from the retinal margin to the
posterior pole from 3 rats for statistical analysis. Every fifth section was counted (50 µm) to
avoid counting the same cell in more than one section; the cells were counted at a ×400
magnification(Wan et al., 2007; Xu et al., 2013).
Statistical analysis
Statistical analyses were performed using SPSS for Windows Version 13.0. Data are described
as mean ± standard error. Statistical comparisons were made using either Student’s two-tailed
t-test or analysis of variance. Differences were considered to be statistically significant at P<
0.05.
Disclosure Statement
The authors declare no competing financial interests.
Acknowledgements
Acknowledgements: This work was supported by the National key Basic Research
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Journal of Cell Science
Program of China. (No. 2013CB967001) and the National Natural Science Foundation of
China (No. 31271400) award to Guang-Hua Peng.
The authors thank Yuxiao Zeng, Qiyou Li and Chuanhuang Weng for assistance with cell
transplantation and ERG examination. The authors declare no competing financial and
material support.
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Fig. 1. C-kit+ cell distribution, isolation and culture. A, Immunofluorescence staining of
C-kit in the human retina. B,D,E, C-kit cells are expressed in the retina and choroid. C, F,
C-kit cells are distributed in the corneoscleral limbus. D, E, Regions in B at higher
magnification. F Regions in C at higher magnification. G, Isolation of C-kit+/SSEA4- cells by
FACS in the upper-left quadrant with depletion of ESCs. H, C-kit+/SSEA4- cells growing in a
monolayer under adherent conditions. J, Formation of neurospheres under non-adherent
conditions after sorting. I,K, Sorted cells were positive for the C-kit receptor by
immunocytochemistry staining and FCS (scale bar: A=200 m, B, C, G, H, I=50m, D, E,
F=10m). OS: ora serrata, INbL: inner neuroblastic layer, ONbL: outer neuroblastic layer.
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Fig. 2. The phenotype of C-kit cells. A, D, G, Negative controls. B, H, Distribution plots of
C-kit+ cells negative for hematopoietic stem cell markers (CD11b and CD45). E, I,
Distribution plots of C-kit+ cells negative for mesenchymal stem cell markers (CD29 and
CD140b). C, F, Distribution plots of C-kit+ cells negative for embryonic stem cell markers
(SSEA4 and MDR [CD243]).
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Fig.
3.
Retinal
progenitor
cell
markers
were
detected
by
FACS
and
immunocytochemistry staining. A-D, More than 90% of C-kit+ cells express retinal
progenitor cell-specific markers and the neural stem cell markers Nestin, RAX, Sox2, and
Pax6, by FACS (n=3 independent experiments). E-H, Representative immunofluorescence
staining for Nestin, Rax, Sox2, and Pax6 (scale bar=50m).
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Fig. 4. Proliferation of C-kit+ cells. A,B, Flow cytometry and immunofluorescence staining
were performed to detect C-kit+ cell proliferation capacity; more than 80% of C-kit cells
expressed ki67. C, Cell cycle distribution of C-kit+ cells was examined; at total of 41.13 ±
2.99% of cells were in the G2 and S phases. D, Growth curve of C-kit cells; a total of
10,000cells/cm2 were plated, and the cell number increased more than 20-fold after 7 days.
(n=3 of independent experiments) (scale bar=50m).
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Fig. 5. The differentiation capacity of C-kit+/SSEA4- cells. A-F, The C-kit+ cells
differentiated into photoreceptor cells, ganglion cells, and glial cells, and expressed
cell-specific markers: Otx2, Crx, recoverin, rhodopsin, the ganglion cell marker Thy1, and the
glial cell marker GFAP (scale bar=50m). G-L, Representative staining of Otx2, Crx,
recoverin, rhodopsin, Thy1, and GFAP were evaluated by FACS.
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Fig. 6. Differentiation, migration, ONL protection of C-kit+ cells in vivo, and ERG
measurements after transplantation. A,E,I, Wild type rat retinas. B,F,J, The retinas from
sham-surgery rats. C,G,K, DiI-labled C-kit+ cells migrated into the inner retina at 4, 8 and 12
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weeks after transplantion and expressed the photoreceptor marker recoverin. D,H,L, Regions
of C,G,K at higher magnification. (A, B, E, F, I, J scale bar=50m, D, H, L scale bar=20m).
M, The ONL of the cell grafted retina was significantly thicker than the control and untreated
groups at 4 weeks (P <0.01), 8 weeks (P <0.01), and 12 weeks (P <0.01). There were no
significant differences in ONL thickness between the control and untreated groups. N-Q, ERG
was performed and b-waves were measured at 4 weeks, 8 weeks, and 12 weeks. There were
significantly high amplitude b-waves in the transplanted group at 4 weeks and 8 weeks
compared to the sham-surgery and untreated groups (P <0.01). However, there was no
significant difference between the sham-surgery and untreated groups after 12 weeks(G). (**P
<0.01) (scale bar=50m).
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Fig. 7. Teratoma assay of C-kit+/SSEA4- cells in SCID mice. A,D, Teratomas were not
observed in the C-kit+/SSEA4- cell group. B,E, Teratoma formation was detected in 1 out of 3
mice at 8 weeks in the hESC group. C, Proportion of C-kit+ cells and hESC cells in the
teratoma assay. F, Teratoma derived from hESC. (scale bar=1 cm)
Figure S1. The description of c-kit+ cell in fetus retina(13 weeks). A, C-kit+ cell
distributed in the inner neuroblastic layer of ora serrate. B, C-kit+ cell distributed in
the inner neuroblastic layer of . C, C-kit+ cell distributed in the optic nerve. Scale bar :
50 μm. RPE: retinal pigment epithelium, ONbL: outer neuroblastic layer, INbL: inner
neuroblastic layer, ON: optic nerve.
Journal of Cell Science | Supplementary Material
Figure S2. The description of c-kit+ cell in fetus cornea(13 weeks). A, description of
c-kit+ cell in fetus cornea. B, regions in A at higher magnification(Scale bar=50μm).
Journal of Cell Science | Supplementary Material