This study developed an in vitro model for differentiating human pluripotent stem cells into retinal neurons. The differentiation process followed identifiable stages of retinal development: 1) Undifferentiated stem cells expressed pluripotent markers. 2) After 10 days of differentiation, eye field populations expressed neural and eye field transcription factors. 3) By day 30, retinal progenitor neurospheres analogous to the optic vesicle expressed retinal progenitor markers. 4) At day 70, the stem cells yielded retinal ganglion cells and photoreceptor cells, demonstrating the ability to derive major retinal cell types.

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Development of an In Vitro Model for the Specification of Retinal Neurons from Human Pluripotent Stem Cells
Kirstin Langer1-2, Akshayalakshmi Sridhar2, and Jason S. Meyer2-4
1Department of Natural and Physical Sciences, Olivet College, 2Department of Biology, Indiana University Purdue University Indianapolis, 3Department of Medical and
Molecular Genetics, Indiana University School of Medicine, 4Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202
Neural Differentiation
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I would like to thank the members of the Meyer lab who have mentored and
assisted in the completion of this study. I would also like to give thanks to
IUPUI and The National Eye Institute for their generous funding that made
this study possible.
Introduction
Undifferentiated hPSCs
Retinal Progenitor Cells
Retinal Ganglion Cells
Photoreceptors
Modeling FASD with hPSCs
Conclusions
AcknowledgementsFig. 1: Characterization of undifferentiated human pluripotent stem cells
(hPSCs) (A) The phenotypic characterization of hPSCs included numerous
colonies of tightly packed cells enclosed by a clearly defined border. (B) RT-
PCR revealed expression of pluripotent markers and the absence of ectoderm,
mesoderm, and endoderm markers. (C-H) Immunocytochemistry illustrated
expression of transcription factors (red) as well as cell surface markers (green)
commonly associated with a state of pluripotency.
Fig. 2: Characterization of retinal eye field populations. (A) After 10 Days of differentiation,
hPSCs displayed a dense, 3D center and a flattened appearance toward the periphery. (B)
RT-PCR indicated the loss of pluripotent markers from 0-10 days of differentiation, along
with the onset of eye field markers. (C-N) Immunocytochemistry indicated widespread
expression of neural and eye field transcription factors.
Human pluripotent stem cells (hPSCs) have progressed into a vital
tool for in vitro studies of human development as well as studying
the progression of diseases affecting specific cell types. hPSCs
have the ability to self renew and give rise to all cell types of the
body1,2. The ability to derive retinal cells from hPSCs allows for in
vitro analysis of these cells and how they may be adversely
affected in the development of the retina3. Thus, the efforts of this
study focused on examining the ability of hPSCs to give rise to
retinal phenotypes, following a predicted, step-wise differentiation
process that closely mimics what is known about retinogenesis.
The differentiation process followed a step-wise progression, with
cells differentiating through identifiable stages of retinal
development yielding retinal neurons, including photoreceptors
and retinal ganglion cells. Subsequently, the ability to utilize
hPSCs to effectively model fetal alcohol spectrum disorders
(FASD) was initiated, as previous studies have documented retinal
defects in zebrafish following ethanol exposure4. Current efforts
are focused upon examining the effects of ethanol exposure on
retinal development from hPSCs, as retinal development is known
to be adversely affected in FASD. The results of these studies
have the potential to serve as a powerful and novel in vitro model
for retinal development as well as disorders affecting the retina. !
References
1. Borooah S. et al., (2013) Using human induced pluripotent stem cells
to treat retinal disease. Prog Retin Eye Res 37: 163-181.
2. Takahashi K. et al., (2006) Induction of pluripotent stem cells from adult
human fibroblast by define factors. Cell 131: 861-872
3. Meyer J. et al., (2009) Modeling early retinal development with human
embyronic and induced pluripotent stem cells PNAS 106: 16698-16703
4. Muralidharan P. et al., (2014) Zebrafish retinal defects induced by
ethanol exposure are rescued by retinoic acid and folic acid supplement.
Alcohol 49: 149-163.
At Day 20, differentiating cells were separated into three
groups, a control, 50mM EtOH, and 75mM EtOH
• From Day 20 to Day 30, retinal differentation
medium was changed every other day as well as the
ethanol treatment for each group.
• At Day 30 ethanol treatment was terminated and the
cells continued to grow in untreated retinal
differentiation medium until Day 60.
Once the cells have differentiated for 60 days, they will be
fixed for cyrostat sectioning and staining
• Staining of cryostat sections will include retinal
ganglion cells and photoreceptor-specific markers to
determine effects of EtOH exposure (if any) between
the control and ethanol treated groups.
Fig. 3: Characterization of retinal progenitor cells and non-retinal forebrain cells. (A) Retinal
neurospheres, indicated by black arrows, were characterized by a bright ring around the outer
edge of the cell. Non-retinal populations, indicated by the white arrows, displayed a larger,
more uniform, and darker phenotype. (B-C) RT-PCR analysis indicated that retinal
neurospheres express neural and retinal makers but did not express forebrain makers, and
also demonstrated increased expression of retinal progenitor markers from 0 to 30 days. (E-
J) Immunocytochemistry analysis displayed the expression of retinal progenitor markers
Chx10 and Pax6 in retinal neurospheres (H) with the absence of forebrain maker Sox1 (F).
Non-retinal neural populations (D) displayed common forebrain neuron features and also
preserved the expression of forebrain and neural makers (E, G). Pluripotent markers, Oct4
and Nanog, were expressed in populations of cells attached to the retinal neurospheres (I-J).
Fig. 5: Characterization of photoreceptors differentiated from hPSCs. (A) RT-PCR analysis
illustrated the expression of photoreceptor associated transcription factors as well as the
expression of phototransduction proteins. (B-G) Immunocytochemistry displayed
photoreceptor-positive cells by the expression of Otx2, Crx, NeuroD1, and Recoverin in
retinal neurospheres.
Fig. 4: Characterization of retinal ganglion cells differentiated from hPSCs. (A) RT-PCR
analysis revealed the expression of the retinal ganglion makers, BRN3, ISLET1, SNCG, and
RBPMS in retinal neurospheres. (B-G) Immunocytochemistry verified the expression of
BRN3-postive cells within huC/D-expressing RGCs, as well as ISLET1-positive ganglion cells
with MAP2-positive extensions in retinal neurospheres.
• The differentiation of hPSCs to retinal neurons follows
a step-wise progression of cells differentiating through
identifiable stages of retinal development.
• Undifferentiated cells expressed pluripotent
transcription factors and cell surface markers.
• Differentiation after 10 days yielded cells of the eye
field, expressing appropriate markers.
• Retinal progenitor neurospheres analogous to the
optic vesicle exhibited appropriate retinal progenitor-
associated makers after 30 days.
• At day 70, hPSCs yielded retinal ganglion cells and
photoreceptor cells, two of the major cells types of the
retina.
C E
D F
Oct4 Nanog
SSEA4 TRA-1-60
Sox2
TRA-1-81
Oct4 Nanog Lin28 Pax6 AFPT
G
H
BA
Day0 Day10Day30
Oct4
Nanog
Otx2
Lhx2
Six3
Six6
Pax6
Chx10
Rax
Pax6FABP7 Rax DLX1 EMX1
A B
C
Chx10/Pax6Sox1/Pax6
BIII Tub/Otx2 Sox1/Pax6
Chx10
Neural Retinal Forebrain
Nanog/Oct4 Chx10/Oct4
D
E
F
G
H
I J
Non-retinal
Non-retinal Non-retinal
Retinal
Retinal Retinal
Retinal
Otx2
Lhx2
Sox1
Pax6
Six6
Rax
Sox2Six3
Otx2
Lhx2
Six6
Six3
Pax6
Rax
Oct4
Nanog
Day 0 Day10
A C D E
F G H
I J
B
K
L M N
Merge
Merge
Merge
Merge
Brn3 huC/D
Islet1 Map2
A
B C D
E
Brn3 Islet1 SNCG RBPMS
Merge
Merge
F G
A
Otx2 Crx
NeuroD1
Crx ND4
Recoverin
Trans. OpsinNRL Arr.
B C D
E F G
Merge
Merge
Overview of Retinal Development
The steps of retinal
d e v e l o p m e n t f r o m
hPSCs yielding retinal
n e u r o n s c a n b e
illustrated at various
s t a g e s i n c l u d i n g
undifferentiated cells
(Day 0), eye field
populations (Day 10),
retinal progenitor cells
(Day 30) and retinal
neurons (Day 70).