Iycee Charles de Gaulle Summary 1.1. in culture, molecular analysis showed expression

1.1. in culture, molecular analysis showed expression

1.1.      Differentiation of human
embryonic stem cells into retinal pigment epithelial cells in feeder- and
serum-free medium

We have developed a new protocol for deriving RPE cells from Relicell®hES1 using direct differentiation method as shown in Fig. 1. Recently,
it had been demonstrated that NIC promoted neural and subsequently RPE
differentiation by preventing apoptosis of neuroectodermal cells (Idelson et al., 2009). In
the first stage of differentiation, we added NIC and IGF-1 to initiate
differentiation towards the neuroectoderm and the eye-field stage. Post 28 days
in culture, molecular analysis showed expression of MiTF while there was no
expression of Rx seen in these cultures. These results suggested that our
combination of growth factors induced RPE progenitors. We next changed the
culture conditions by addition of APRE-19-conditioned medium for maturation of
these progenitors to form RPE cells. ARPE-19 is a spontaneously arising human
RPE cell line with normal karyology and has structural and functional
properties characteristic of RPE cells in
vivo. When cultured in the presence of conditioned medium from ARPE-19
cells, these RPE progenitors started forming RPE-like cell clusters in culture
by day 49. They appeared as pigmented cell clusters under phase-contrast
microscope. Later, these cells accumulated more pigmentation and adopted a
typical hexagonal morphology with a squamous appearance (Fig. 2A). Further,
hESC-derived RPE cells formed prominent colonies of pigmented cells visible
with the naked eye beyond 100 days in culture (Fig. 2B).

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1.2.      Gene expression and
immunofluorescence study of hESC-derived RPE cells

We performed semi-quantitative RT-PCR for known markers at different
stages of differentiation (day 0, 14, 28, 60, and 100) in Relicell®hES1
and compared their expression levels with adult human retinal RNA and ARPE-19
(A-19) cell line (positive control), and UCMSCs (negative control). Our results
demonstrated that Relicell®hES1 expressed Oct-4 on day 0 and as
differentiation was initiated there was a loss of Oct-4 expression (day 14, 28,
60, and 100).  We also observed that both
neuroectodermal and early eye-field markers, Nestin and Pax6, were expressed
throughout the differentiation stages. Early neural-retina marker, Rx, showed
very faint expression by day 14 and disappeared thereafter. Expression of Chx10
was seen by day 14 and up to day 28 that was later down-regulated as these
cells showed commitment towards RPE cell fate. The early RPE marker, MiTF, was
detected as early as day 14 and the expression increased during differentiation
(Fig. 3A). The RPE cell-specific markers, RPE65, CRALBP1, and Bestrophin, were
detected for the first time at day 60 and the expression of RPE65 increased by
day 100 in Relicell®hES1. Other RPE cell markers, Otx2,
PEDF, PMEL17, MERTK, and VEGF?A were expressed relatively early during the
differentiation with TYRP1 being expressed by day 60. The mesodermal marker,
Brachyury, and the endodermal marker, GATA-4, were not expressed during any of
the stages of differentiation. In addition, the early and late markers of
photoreceptor differentiation, CRX, Recoverin, Opsin, and Rhodopsin, were
absent in both these cells strongly suggesting the RPE cell fate of the derived
cells (Fig. 3B). Of the studied genes, undifferentiated cells showed the
expression of Nestin, Pax6, Otx2, PEDF, PMEL17, and VEGF-A; the undifferentiated
UCMSCs used as negative control also showed expression of PEDF and VEGF-A.


1.3.      Electron microscopic analysis
of hESC-derived RPE cells

For any application of these derived cells, it is imperative that
the cells possess functional capabilities. Electron microscopic analysis of
hESC-derived RPE cells at day 100 (post-differentiation) was done to determine
whether the generated RPE cells have ultrastructural characteristics of the
RPE. The cells were highly polarized with basally located nuclei; presence of
well-developed tight-junction complexes; and apical microvilli (Fig. 4).
Melanosomes responsible for pigmentation normally present in RPE cells were
clearly observed within the hESC-derived RPE cells.

Secretion of trophic factors viz.,
VEGF-A and PEDF were analyzed by ELISA in cell supernatants collected at 48 h
time interval. Results showed that hESC-derived RPE cells secreted significant
levels of VEGF-A and PEDF into the culture supernatant as compared to the
undifferentiated hESCs (Data not shown).


Expansion and growth of differentiated RPE cells

We have shown that hESCs can be efficiently differentiated to form
clusters of RPE that are tightly packed with cells. These cells show typical
hexagonal morphology and dense pigmentation in culture. One of the questions
addressed was whether we can expand these cells to larger numbers suitable for
regenerative cell therapy. In the first attempts, we dissociated these RPE
clusters to single cells with various methods (Supplementary information, Table
S2). However, these techniques did not help as either the cell clusters did not
detach or there was a loss of RPE morphology.

Since none of the methods yielded satisfactory results,
we attempted to remove cells that were around RPE clusters. Using TrypLETM
treatment, we dissociated the cells surrounding the RPE clusters and removed
them from the dish. The RPE clusters were then maintained in culture for a
period of 7 days in DMEM supplemented with B27 medium. There was no change
observed in the morphology of RPE clusters. We treated theses clusters with reduced
serum and cocktail of growth factors (PDGF-CC, rhEGF, bFGF) thereafter and it
was interesting to note that the cells started budding out of the clusters
between days 4 and 7 and formed a monolayer of cells in culture by day 15.
These RPE cells were further dissociated with TrypLETM and further
expanded and matured in culture multiple times.


1.5.      Maturation of expanded
hESC-derived RPE cells

Since we could now expand hESC-derived RPE cells, we investigated if
these expanded cells could be made to regain hexagonal RPE morphology. Human
ESC?derived RPE cells were first seeded on MatrigelTM and cultured
for 6-7 days in DBS-PEF medium. The cells expanded in these conditions but did
not show any signs of pigmentation or hexagonal morphology normally visible in
RPE cells. It has been shown by various investigators that upon withdrawal of
bFGF, RPE cells can spontaneously differentiate into pigmented cells forming
typical RPE cell morphology (Klimanskaya et al., 2004). Upon reaching
confluence, growth factors were withdrawn and the cells were further incubated
in DB medium.  Within one week after
withdrawal of growth factors, typical RPE monolayers with mild pigmentation was
observed in culture (Fig. 5) indicating that it is possible to regain RPE
morphology and pigmentation in the expanded hESC-derived cells. At this time,
they were designated as expanded and matured RPE (ExMat-RPE) cells. Further, hESC-derived RPE cells cultured on
MatrigelTM developed heavy pigmentation by day 21 that was visible
to the naked eye, in contrast to those grown on tissue culture treated plastic surface
(Fig. 6).


1.6.      Gene expression analysis of ExMat-RPE cells

ExMat-RPE cells were characterized to confirm RPE maturation. Expression
of specific markers associated with the cellular function of the mature RPE was
investigated by RT-PCR. Transcript expression levels of early neural marker,
Nestin; eye-field marker, Pax6; early RPE marker, MiTF; mature-RPE markers,
RPE65, CRALBP1, and Bestrophin; and melanogenic marker, TYRP1 were specifically
detected in ExMat-RPE cells. Other
RPE cell markers such as Otx2; neurotrophic factor, PEDF; phagocytic marker,
MERTK; immature melanosome marker, PMEL17; and VEGF-A were also expressed in
these cells. The pluripotent stem cell marker, Oct-4 was expressed in the
undifferentiated hESCs and was not detected in ExMat-RPE cells. The mesodermal marker, Brachyury; the endodermal
marker, GATA-4; early neural-retinal progenitor marker, Chx10, was not
expressed in these cells. In addition, the early and late markers of
photoreceptor differentiation, CRX, Recoverin, Opsin, and Rhodopsin, were also
absent in these cells as expected. Adult retinal RNA was used as positive control
(Fig. 7).


1.7.      Characterization of ExMat-RPE cells by
immunofluorescence and flow cytometry

Immunostaining of the ExMat-RPE
showed the expression of the early neural and eye-field markers, Nestin, Pax6,
and MiTF; and mature-RPE markers,
RPE65, CRALBP1, Bestrophin, Ezrin, CK18, and the tight-junction protein,
ZO-1 (Fig. 8). Importantly, pigmented hESC-derived RPE cells did not express
the pluripotent stem cell marker, Oct-4 but showed weak expression of SSEA-4
indicating that the mature cells resembled adult RPE cells (Fig. 9).


1.8.      Functional analysis of the ExMat-RPE cells

The functionality of ExMat-RPE
cells was shown by electron microscopy, phagocytosis, and polarized secretion
of trophic factors. Electron micrographs showed typical features characteristic
of mature RPE that included pigmented cuboidal epithelial cells with apical
microvilli, abundant presence of melanin granules, and tight junctions between
the cells (Fig. 10).

To simulate in vivo
conditions in vitro, we used newly
introduced pHrodoTM BioParticles® red conjugate to assess
the functional potential of these mature RPE cells. Immunofluorescence
microscopy of cells immunostained with FITC-phalloidin showed the
internalization of fluorescently-labelled red pHrodoTM BioParticles®
(Fig. 11).

Polarized secretion of trophic factors was seen in ExMat-RPE cells. In this experiment, the
culture supernatants were collected from the apical and the basolateral chamber
of the porous Transwell insert (Supplementary information, Fig. S1). The mean
(± SEM) concentration of VEGF-A in the apical and basolateral supernatants
was 4.6 ± 0.08 ng/ml and 10.5 ± 0.09 ng/ml, respectively (Fig. 12A) while,
the mean (± SEM) concentration of PEDF in the apical and basolateral
supernatants was 189.5 ± 0.7 ng/ml and 196.5 ± 0.5 ng/ml, respectively (Fig.
12B). ExMat-RPE cells grown and
matured on Transwell culture membranes preferentially secreted VEGF-A to the
basolateral side while no significant difference in the secretion of PEDF was
noted in these cultures.


1.9.      Expression of
co-stimulatory molecules by ExMat-RPE cells in the absence and presence of

In order to utilize hESC-derived RPE cells for allogeneic
transplantation, it is very important to study the expression pattern of
co-stimulatory/ immunoregulatory molecules expressed by these cells. We
performed gene expression studies on the ExMat-RPE
cells that were untreated and treated with 10 ng/ml IFN-? for 5 days (Fig. 13A).
ExMat-RPE cells showed B7-H1, B7-DC,
IDO, HLA-G, and CD86 mRNA expression but no expression of HLA-DR and CD80 genes
were observed (Fig. 13B). However, mRNA expression corresponding to HLA-DR
was detected following IFN-? treatment for 5 days. IFN-? treatment of these
cells showed further increase in the mRNA levels of B7-H1, B7-DC, and IDO while
there was no change in the mRNA expression of HLA-G and CD86. The data obtained
from one representative experiment are shown in Fig. 13B.

Next, immunophenotypic analysis for the expression of
co-stimulatory/ immunoregulatory proteins was also performed on ExMat-RPE cells. In comparison to
control cells, a significant increase in the expression of HLA-DR (58.3% ±
2.3%), B7-H1 (78.0% ± 3.1%), IDO (99.1% ± 2.9%), and HLA-ABC (84.1% 3.6%) was
noted on day 5 after IFN?? treatment. As compared to ARPE-19 cells, IFN-? did
not induce expression of B7?DC, HLA-G, CD80, and CD86 molecules in ExMat-RPE cells. Representative results
are shown in Fig. 13C.


1.10.  Effect of ExMat-RPE cells on allogeneic
T cell proliferation in vitro

Purified allogeneic CD3+ T cells isolated from healthy
donors were labelled with Carboxyfluorescein diacetate N-Succinimidyl Ester (CFSE)
dye and co-cultured with ExMat-RPE
cells in vitro for a period of 5 days
in a mixed lymphocyte reaction (MLR) study as previously described (Vasania et al., 2011). A
decrease in the fluorescence intensity of CFSE was considered as an indication
of T cell proliferation. ExMat-RPE
cells failed to elicit CD4+ T cell proliferation after 5 days of culture
(Fig. 14). These cells retained maximum levels of CFSE dye (Fig. 14C) at an
intensity comparable to that of T cells that were cultured alone (Fig. 14A)
suggesting that the allogeneic T cells did not proliferate in the presence of ExMat-RPE cells during the 5 day culture
period. Monocyte-derived dendritic cells (mDCs) were used as positive control
and, as expected, mDCs induced 45.0% of CD4+ T cell proliferation in
the same assay (Fig. 14B). ExMat-RPE
cells, interestingly, did not induce T cell proliferation even after exposing
the cells to IFN-? (Fig. 14D). These results indicate that ExMat-RPE cells are unable to induce allogeneic CD4+
T cell proliferation in the presence of IFN?? and after upregulation of
HLA-DR antigen as shown earlier.