Age-related sheets of human fetal retinal progenitor cells together

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly
patients with approximately 25 to 30 million people affected worldwide by some
form of this disease (Chopdar
et al., 2003; Tasman and Rovner, 2004). The retinal pigment epithelium is
a monolayer of pigmented hexagonal cells stationed between the photoreceptor
and the choroid layers. It plays important roles in the maintenance and
function of the retina and its neighboring photoreceptors. Degeneration or
dysfunction of RPE leads to blindness through loss of photoreceptors. A variety
of therapeutic approaches to delay the degenerative process is being developed,
but the truth is that many patients sooner or later lose their sight (Idelson et al., 2009).
Since 1986, attempts have been made to transplant sheets of human fetal retinal
progenitor cells together with RPE to the sub-retinal space to replace both
lost photoreceptors and RPE, and demonstrated efficacy in both animals and
humans (Radtke et al.,
2008). However, there are both ethical and supply issues with the use of
fetal tissue. Most other approaches are restricted to the rescue of endogenous
retinal cells, which do not restore lost function and is limited to the early
stages of disease. However, in animal models of retinal degeneration,
photoreceptor rescue and preservation of visual function have been demonstrated
after sub-retinal transplantation of RPE cells (Little et al., 1996; Sauvé et al., 2002). Even partial
restoration of visual function was reported in humans after autologous transplantation
of RPE layer (da Cruz et al., 2007). Recently, clinical trials using RPE cells derived from pluripotent
stem cells are being conducted worldwide to establish the safety and survival
of the transplanted cells (Kimbrel and Lanza, 2015). Two phase I/II studies have already shown
promising outcome in patients with age-related macular degeneration and
Stargardt’s macular dystrophy (Schwartz et al., 2015).

Human ESCs have been thought to be
a promising source of replacement cells for regenerative medicine since their
discovery in 1998 (Thomson et al., 1998). Human ESCs derived from human blastocysts maintain pluripotency,
proliferative potential and karyotypic stability for prolonged periods.
Interestingly, these pluripotent stem cells not only have the capability to
provide an unlimited supply of cells but also can be directed in vitro to a desired cell type such as
RPE cells. Initial reports investigating spontaneous hESC differentiation into
RPE-like cells upon withdrawal of basic fibroblast growth factor (bFGF)
generated limited number of pigment-producing RPE cells. Subsequently, several
laboratories have generated RPE-like cells from human embryonic stem cells using
more defined culture conditions and holds great potential for the treatment of
retinal diseases (Carr et al., 2009; Meyer et al.,
2009; Osakada et al., 2009a). More recently, Idelson
et al. (2011) reported the generation of RPE-like cells using
nicotinamide (NIC) and activin A. In this report, we present a multistep
differentiation protocol using B27, NIC, insulin-like growth factor-1 (IGF-1),
and ARPE-19 conditioned medium to derive pigmented cluster of RPE cells in
feeder-free conditions. In order to utilize the derived RPE cells for clinical
transplantation purposes, it was important for us to determine the expansion
potential of these cells. Using serum and combination of growth factors, it was
possible to expand RPE cells from clusters of
RPE. The expanded cells maintained karyotypic stability in vitro. Withdrawal of growth factors resulted in maturation
and gaining pigmentation and polygonal morphology in these cells which were
termed as ExMat-RPE cells. These
cells expressed several mature RPE markers and possessed RPE cell characteristics,
such as presence of apical microvilli, abundant melanosomes and tight
junctions. They were able to phagocytose fluorescent labeled bioparticles and
also secrete trophic factors like vascular endothelial growth factor-A (VEGF-A)
and pigment epithelium-derived factor (PEDF).

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In addition, determining the
immunogenicity of ExMat-RPE cells
would be critical for successful transplantation and long-term survival of the
transplanted cells. Recently, the first step towards transplanting hESC-derived
RPE cells into patients with AMD and Stargardt’s macular dystrophy have given
promising results by demonstrating the safety benefits. However, in vitro immunological study on hESC?derived
RPE cells have not been reported. Recently, we demonstrated the unique
immunological properties of expanded and cryopreserved human limbal epithelial
stem cells (hLESCs) and thought it was also important to determine the
immunological properties of ExMat-RPE
cells in vitro as well. It has been
shown by various investigators including us that molecular homologues of
B7-like ligands, such as B7-H1 and B7-DC, indoleamine 2,3-dioxygenase (IDO),
and a non-classical human leukocyte antigen (HLA) class I molecule, HLA-G, are
known to be expressed by various ocular cell types.

In the present study, we report
that functional ExMat-RPE cells can
be efficiently derived and expanded from RPE-like clusters using appropriate
culture conditions. We also report for the first time that ExMat-RPE cells express negative immunoregulatory molecule, IDO, and
induce expression of B7-H1 in the presence of interferon-gamma (IFN-?).
Further, these cells were also unable to induce T cell proliferation in vitro. Collectively, these results
suggest that ExMat-RPE cells can possibly
survive within the allogeneic environment and in future can be used for
treating retinal diseases.