Ophthalmic Res 2012;48(suppl 1):1–5
DOI: 10.1159/000339839
Published online: August 21, 2012
In vitro Differentiation of Adult Adipose
Mesenchymal Stem Cells into Retinal
Progenitor Cells
Gustavo A. Moviglia a Nahuel Blasetti a Jorge O. Zarate b David E. Pelayes b
a
Center for Research in Tissue Engineering and Cellular Therapies and b Center for Applied Research and High
Complexity in Ophthalmology, Maimónides University, Buenos Aires, Argentina
Key Words
Stem cells ⴢ Retinal progenitor cells ⴢ Adult mesenchymal
stem cells
Abstract
Introduction: It has been previously shown that adult mesenchymal stem cells (MSCs) differentiate into neural progenitor cells (NPCs) and that the differentiation process was
completed in 24–48 h. In this previous study, MSCs from a
bone marrow or fat source were co-incubated with homologous autoaggressive cells (ECs) against nerve tissue, and
these NPCs were successfully used in human regenerative
therapeutic approaches. The present study was conducted
to investigate whether a similar differentiation method
could be used to obtain autologous retinal progenitor cells
(RPCs). Methods: Human Th1 cells against retinal tissue were
obtained by challenging human blood mononuclear cells
with an eye lysate of bovine origin; negative selection was
performed using a specific immunomagnetic bead cocktail.
Fat MSCs were obtained from a human donor through mechanical and enzymatic dissociation of a surgical sample.
The ECs and MSCs were co-cultured in a serum-free medium
without the addition of cytokines for 0, 24, 48 and 72 h. The
plastic adherent cells were morphologically examined using
inverted-phase microscopy and characterized by immunofluorescent staining using antibodies against Pax 6, TUBB3,
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GFAP, Bestrophin 2, RPE 65, OPN1 SW, and rhodopsin antigens. Results: The early signs of MSC differentiation into
RPCs were observed at 24 h of co-culture, and the early differentiated retinal linage cells appeared at 72 h (neurons,
rods, Müller cells, retinal ganglion cells and retinal pigmented epithelial cells). These changes increased during further
culture. Conclusion: The results reported here support the
development of a method to obtain a large number of autologous adult RPCs, which could be used to treat different
retinopathies.
Copyright © 2012 S. Karger AG, Basel
Introduction
According to Baker and Brown [1]:
Researchers have demonstrated that stem-cell transplants can
survive, migrate, differentiate, and integrate within the retina.
Stem cells from various developmental stages have been used in
these experiments, including embryonic stem cells (MSC), neural
stem cells (NSC), mesenchymal stem cells, retinal stem cells, and
adult stem cells from the ciliary margin. Not only can these transplants adopt retina-like morphologies and phenotypes, but they
have also shown evidence of synaptic reconnection and visual recovery in both animal and human studies. Still, work must be
performed to achieve higher yields of functioning retinal neurons
and to promote better integration within the host retina.
David E. Pelayes, MD, PhD
Castro Barros 321
Ciudad Autónoma Buenos Aires C1178AAG (Argentina)
Tel. +54 11 4432 8696
E-Mail davidpelayes @ gmail.com
Although the autologous adult source of no genetically manipulated stem cells has lower potential to induce
autoimmunity or neoplasia [2], the production and/or
differentiation of retinal progenitor cells (RPCs) remain
the main obstacles for their use [1, 3, 4].
In a previous work, we have demonstrated that mesenchymal stem cells (MSCs) from adult bone marrow sources may be differentiated into neural stem cells [5]. Moreover, these cells have been shown to be useful in the treatment of chronic spinal cord injury patients [6, 7]. Such a
treatment has the advantage of being fast, safe and does
not require the supplementation of differentiation factors.
During the first 48 h of culture, immune effector cells
(ECs) against different spinal cord antigens induce the
differentiation of autologous MSCs into neuroblasts [5].
Because the MSCs from adult adipose tissue have shown
a tremendous proliferative power, we investigated the
possibility of improving the yield of adult retina stem cell
production through in vitro experiments [9, 10]. The
present report summarizes the main results that prove
our hypothesis.
Materials and Methods
Source of Cells
Human ECs against retinal antigens and human fat MSCs
have been obtained from donors; written, informed consent was
obtained, and the study was approved by the Maimonides Ethics
Committee.
EC Purification
The donors underwent an apheresis process using a Cobe
Spectra (Gambro, Chicago, Ill., USA) cell separator to obtain the
buffy coat; approximately 2 blood volumes from each donor were
processed. The obtained buffy coat suspension had a composition
of 85% mononuclear cells (MNCs), 1.8 ! 106/␮l red blood cells,
and 6 ! 105/␮l platelets. This MNC sample was purified using a
Ficoll-Hypaque gradient (1.077 density), and the obtained cells
were washed in DBSS without Ca++ or Mg++. The composition of
the fraction was approximately 98% MNCs, 0.2 ! 106/␮l red
blood cells, and 1 ! 104/ml platelets. The sample was not used if
the obtained buffy coat or the concentrated MNCs did not meet
the above-mentioned standards.
The MNC suspension was cultured for 4 days in DMEM enriched with a partial bovine eye hydrolization (Laboratorios Villar, Rosario, Santa Fe, Argentina).
On day 5, the CD3+ lymphocytes were isolated by negative selection using a monoclonal antibody cocktail against the undesired cells (monoclonal antibodies against CD14, CD16, CD19,
CD56 and glycophorin A). This first incubation was followed by
a second incubation with a solution of antibodies attached to
paramagnetic Teflon beads (Stem Sep kit, Stem Cell Technology,
Vancouver, B.C., Canada). After immuno-labelling the cell suspension, these lymphocytes were passed through a powerful mag-
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Ophthalmic Res 2012;48(suppl 1):1–5
netic field, which allowed the passage of the CD3+ cells and retained the remainder of the cells. The resulting CD3+ cell fraction
was enriched up to 96%.
This CD3+-enriched suspension was then labelled with an anti-CD25 monoclonal antibody solution (Stem Sep kit), and the
cells were attached to paramagnetic Teflon beads for a second selection. The proportion of the cell population phenotypes, CD3+
CD25– and CD3+ CD25+ varied according to the donor.
MSC Purification and Expansion
In an operating room, a surgeon performed a dermolipectomy
to procure 50–200 g of adipose tissue from a donor.
In the GMP facility of Maimonides University, the fat tissue
was mechanically and enzymatically dissociated (collagenase
type IV 100 ␮g/ml) to obtain a single MNC suspension. Both free
fat cells and free fat droplets were obtained, and the fat droplets
were eliminated by discarding the supernatant. The free fat cells
were cultured in serum-free Mesencult Medium (Stem Cell Technology) for 24 h. The non-adherent cells were discarded, and the
adhered cells were cultured until a semi-confluent stage and then
re-seeded and cultured during 3 weeks. The culture conditions
were 37 ° C in an atmosphere of 95% O2 and 5% CO2 using a Thermo Forma쏐 CO2 incubator.
The culture was monitored using a phase-contrast inverted
microscope (Nikon Eclipse TS 100).
Co-Culture of Effector T Cells and MSCs and the MSC and
RPC Characterization
The adherent MSCs were harvested and seeded on 25-mm2
6-well plastic plates. The cells were then co-incubated either with
peripheral MNCs or with CD3+ CD25– lymphocytes against retinal antigens. The tissue culture medium was serum- and cytokine-free, and the cells were co-incubated for 0, 24, 48 and 72 h.
The morphology of the cells attached to the plastic were examined
using a phase-contrast inverted microscope (Nikon Eclipse TS
100) and characterized by immuno-staining for the following:
Pax6, a marker of RPC (Santa Cruz Biotechnology); TUBB3, a
marker of young neurons (Sigma); Bestrophin 2, a marker of basal plasma membrane of non-pigmented epithelium (Millipore);
glial fibrillary acidic protein (GFAP), a marker of astrocytes and
Müller cells (Sigma); RPE 65, a marker of retinal pigmented epithelium (Millipore); OPN1 SW, a marker of cones (Millipore), and
rhodopsin, a marker of rods (Santa Cruz Biotechnology).
Results
The identities of MSC and RPC cell suspensions were
mainly based on the cytological appearance of the tissue
culture by observation using phase-contrast inverted microscopy. At least 90% of the MSCs observed in a field of
view (magnification !10) should have a homogeneous
cytoplasm, with at least 4 nucleoli per nucleus and a light
vacuolization pattern surrounding the nucleolus (fig. 1a).
After 24 h of co-culture, 80% of the observed cells exhibited perinuclear cytoplasmic granules and a single
large nucleus with no more than 2 prominent nucleoli
Moviglia /Blasetti /Zarate /Pelayes
Color version available online
Fig. 1. Phase contrast morphology of the
cultured cells. a Non-stimulated MSC.
b After 24 h of co-culture it is possible to
observe cells with the shape of RPCs.
c, d After 72 h of co-culture cells with the
shape of retinal ganglion cells (RGC),
Müller cells (MC), retinal pigmented epithelium cells (RPE) and rods can be observed.
a
b
c
d
(fig. 1b). The neu 66 marker identified these cells as
RPCs.
These changes increased during the following observation points (fig. 1c, d). At 72 h after co-culture, the morphological and immunological analyses of the co-cultured MSCs revealed early differentiation patterns of the
RPCs and early differentiated cell lines (neurons, rods,
Müller cells, retinal ganglion cells and retinal pigment
epithelium cells).
The immune stain and ulterior analysis with confocal
microscopy confirmed our phase contrast observations.
The main results are summarized in figure 2. At 72 h
there is an important number of cells that mark positive
for GFAP and Bestrophin 2 that may be interpreted as
common progenitor cells for both non-pigmented epithelium cells and Müller cells (fig. 2a–c). There are also several cells that have a positive marker for tubulin beta III,
that is indicative of their young neuron progeny (fig. 2d),
and some of them are positive for OPN1 SW, that is a
marker for cone cells (fig. 2e).
Discussion
One of the most promising therapies to repair retinal
degeneration and retinal trauma lesions is based on the
use of stem cells [1, 3].
In vitro Differentiation of Adult Adipose
MSCs into RPCs
Several authors have shown in animals that some adult
cells (MSCs and retinal pigmented epithelium cells) have
the potential to differentiate into RPCs under in vitro and
in vivo conditions, yet the small amount of differentiation and in vitro growth limits their use [1, 4, 5, 10].
In contrast, human embryonic stem cells and induced
pluripotent stem cells [11, 12] present significant differentiation and growth in vitro. However, according to previous reports and the official statement of the European
Medicine Agency [2], human embryonic stem cells and
induced pluripotent stem cells may induce autoimmunity
or tumour growth. The same report states that autologous
adult stem cells have proved to be safe and pose a very low
risk to induce autoimmunity and carcinogenesis [2].
The present study shows that fat-derived MSCs may be
differentiated into RPCs using co-culture with ECs. The
morphological phase-contrast pattern observed in these
cells (nuclear and cytoplasmic changes) indicates differentiation, which is supported by the specific immunofluorescent staining of specific cell lineage markers that match the
different morphological changes [13, 14]. A similar result
was previously described by us for the differentiation of
adult bone marrow-derived MSCs into neuroblasts.
This differentiation method of adult MSCs into RPCs
appears to be a valid way to overcome both the safety concern and problems with production. In effect, MSCs,
even from the elderly, may be cultured in large quantities
Ophthalmic Res 2012;48(suppl 1):1–5
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Color version available online
a
c
b
e
d
Fig. 2. Confocal microscopy analysis of the cultured cells. a GFAP-positive cells. b Bestrophin 2-positive cells.
c Merge image of a and b. d Tubulin beta III-positive cells. e OPN1 SW-positive cells.
using defined tissue culture media. ECs may be obtained
from the same donor (or patient), generating a yield of at
least 80% of differentiated RPCs, which may, in turn, differentiate into the different progeny of retinal cells [9].
According to Moalem et al. [15], this differentiation
induction has been attributed to the specific protective
action of the tissue-specific autoimmune cells. This action may also be related to the production of neurotrophins by the anti-optic nerve cells, as described by Barouch
and Schwartz [16].
The short duration of this described differentiation
process was in contrast to the relatively long period of
time necessary to induce RPCs from either human embryonic stem cells or induced pluripotent stem cells. We
attribute the fast rate to the fact that the activated spe-
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of the cross-talk between the MSCs and ECs.
These promising results have prompted us to develop
a therapeutic approach similar to the one that was described for chronic spinal cord injury [7, 8].
Disclosure Statement
Proprietary interests: none. Grants: none. Ethical statement:
this study complies with the Declaration of Helsinki including
current revisions and the Good Clinical Practice guidelines. The
procedures followed were in accordance with institutional guidelines and all subjects gave written informed consent before the
study. Financial disclosure: none.
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