Induced pluripotent stem cells

1. Modeling Early Retinal Development with Human Embryonic and Induced Pluripotent Stem
Cells
Author(s): Jason S. Meyer, Rebecca L. Shearer, Elizabeth E. Capowski, Lynda S. Wright, Kyle
A. Wallace, Erin L. Mcmillan, Su-chun Zhang, David M. Gamm, James Thomsan
Reviewed work(s):
Source: Proceedings of the National Academy of Sciences of the United States of America,
Vol. 106, No. 39 (Sep. 29, 2009), pp. 16698-16703
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/40484967 .
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2. earlyretinal
Modeling development withhuman
embryonic induced
and stem
pluripotent cells
Jason S. Meyer3,Rebecca L Shearer3, Elizabeth E. Capowski8, Lynda S. Wright8,
Kyle A. Wallace3, ErinL McMillan8,
Su-ChunZhang3b, and David M. Gamma'cd'1
aStem Cell Research Program,Waisman Center,bDepartmentsof Anatomy and Neurology,department of Ophthalmology and Visual Sciences, and dEye
1500 Highland Avenue, University Wisconsin-Madison,Madison Wl 53705
Research Institute, of
Edited by JamesThomson, University Wisconsin,Madison, Wl, and approved July 2009 (received for review May 15, 2009)
of 23,
Human pluripotent stem cells have the potentialto provide compre- and maturation follows sequenceand timecourse highly
a
hensivemodel systemsforthe earlieststages of human ontogenesis. reminiscent normal
of retinal
development. Furthermore, the
To serve in this capacity, these cells must undergo a targeted, process retinal
of differentiation be selectively
could altered via
stepwise differentiation process that follows a normaldevelopmen- of
manipulation endogenous developmental signaling
pathways.
tal timeline.Here we demonstratethe abilityof both human embry- We theninvestigated whether same culture
the method was
onicstemcells(hESCs)and inducedpluripotent stem(iPS) cellsto meet capable ofgenerating identical
an cohort developing
of retinal
these requirements human retinogenesis.Upon differentiation, celltypes
for from human induced stem a
pluripotent cells, recently
hESCsinitially yieldeda highly enrichedpopulation of earlyeye field described sourceof pluripotent cells derived
stem from skin
cells. Thereafter, subset of cells acquired features of advancing
a fibroblasts 11). Cell populations
(10, expressing morphologic
retinal differentiation a sequence and timecourse that mimicked
in in features and/or markers theeyefield,
of retinalpigment epithe-
vivo human retinaldevelopment.Applicationof this culturemethod lium,neural retinalprogenitors, photoreceptorprecursors,and
to a human iPS cell line also generated retina-specific types at
cell
photoreceptors observed differentiating iPS cell
were in human
comparable times in vitro. Lastly,altering endogenous signaling cultures time
at points predicted results
by using hESCs.These
duringdifferentiation affectedlineage-specific gene expression in a findings support roleforhuman
a pluripotent cellsas in
stem
mannerconsistentwith established mechanismsof early neural and vitromodel to
systems investigate mechanisms involved retinal
in
retinal cell fate determination.These findings should aid in the anddifferentiationofindividual retinal types.
cell
specification
investigation the molecularevents governingretinalspecification
of
fromhuman pluripotent stem cells. Results
Eye Specification Human
Field from Stem The
Embryonic Cells. appear-
study human
of development is limited a lackofmodel anceofeyefield
by cellswithin primitive anterior neuroepithelium
systems canreproduce precise
that the and of
sequence timing is thefirst phaseinthestepwise production a retinal
of pheno-
cellular molecular
and events occurduring
that human embryo- type from undifferentiated
an pluripotent cell(12,13)(Fig.
stem
genesis, organogénesis, tissue
and differentiation. However, the L4). Previous reports have demonstrated hESC-derived
that
advent human
of pluripotent celltechnology
stem affords a unique neuroectodermal willadoptanterior
cells neuroepithelial char-
opportunity to follow full of cell
the course lineage-specific pro- acteristics theabsence exogenous in of signaling molecules (14,
duction vitro 2). The retina
in (1, an
provides optimal system to 15).In thecurrent study (Fig.'B), hESCswere differentiated as
investigate potential to itswell-defined conserved free-floating
this due and hESC aggregates and prompted adhereto
to
developmental program theavailabilitymarkers distin- laminin-coated
and of to culture dishes permit
to neuralrosette forma-
guish eachmajor of
stage early retinogenesis. In addition, human tion. 16 of
After days differentiation, rosette-containing colonies
embryonic cells
stem (hESCs)displaypropensitya toproduce cells were to as
removed grow neurospheres. During this
mechanically
with retinal characteristics Onecriterion assessing
(3,4). for hESC- hESCsrapidly expression thepluripotency
lost of
process, genes
based model is to
developmental systemsthecapacity recapitulate Oct4andNanog acquired and of factors
in the embryo a associated
in expression transcription
the normal maturation sequencepresent with field
eye specification Six3,
(Rx, Sixò, Lhx2, Til),
controlled, stepwise fashion such
(1,2). Preferably, systems should anterior neural induction
of
to test effects developmental
the
neural specification (Otx2)andgeneral
the
alsoprovide opportunity
for cell (Pax6,Soxl,Sox2)(Fig.1C). In RT-PCRexperiments, was Pax6
stimuli enrich early populations,
and thereby reducing present a doublet
cell
unidentified lineages.
as band, reflecting expression both
the of the
contamination undesired
from and/or
To date,hESC studies have focused the derivation
on Pax6(-5a) and Pax6(+5a) isoforms. appropriate
of and
The staging
lineage this of early population further
cell was supported by
subsets retinal populations, emphasis thepro- theabsenceof the
of cell with on
duction either
of retinal ormore mature cells photoreceptor precursor-specific transcrip-
progenitors 6)
(5, tion factor Crx and the spinalcord-associated transcription
such retinal
as pigment epithelium (RPE) (3, 4) or photorecep- factor HoxB4,as wellas markers other of germ layers suchas
tors Many these
(7). of studies various
used exogenous factors to
within brachyury and
(mesoderm) alpha-fetoprotein (endoderm). Im-
the
increase percentage early of cell
retinal types present showedthatnearly cells within
all these
the mixed of hESCs.
population differentiating However, despite munocytochemistry
these recent advances, ability hESCsto produce highly
the of a
enriched population cells at the earliest
of stage of retinal Author contributions:J.S.M.,E.E.C., and D.M.G. designed research; J.S.M.,R.L.S.,K.A.W.,
specification can
that progress through each of thekeydevel- and E.L.M. performed research; S.-C.Z. contributed new reagents/analytictools; J.S.M.,
opmental stagesof the retinahas yet to be demonstrated. R.L.S., E.E.C., L.S.W., K.A.W., S.-C.Z., and D.M.G. analyzed data; and J.S.M.,L.S.W., and
Moreover, timing onsetof selectedstagesin retinal D.M.G. wrote paper.
the of the
development varied
has widely among published human pluri- The authors declare no conflictof interest.
potent stemcell differentiation protocols, noneof which ap- This article is a PNAS DirectSubmission.
proximated timeline normal
the of human retinogenesis (5-9). See Commentaryon page 16543.
We addressed these issues first examining major
by each step 1To whom correspondence should be addressed. E-mail:dgamm@wisc.edu.
in thedevelopment definitive
of cell
retinal populations from This article contains supporting informationonline at www.pnas.org/cgi/content/full/
hESCs.In doing wedemonstrated cellfate
so, that specification 0905245106/DCSupplemental.
16698-16703 | PNAS | September29, 2009 | vol.106 | no. 39 www.pnas.org/cgi/doi/10.1073/pnas.0905245106

3. <
■z.
!E
o
u
h
¡i
s
Fig. 2. Highly derivation eye fieldphenotypes
efficient of from hESCs. RT-PCR
(A)
analysisshowingthe onsetof Pax6and Rxgene expression concomitant of
and loss
Oct4. (B and 0 qPCR analysis Oct4 gene expression and Pax6 and Rxgene
of (8)
expression Values were expressedas foldchange relative undifferentiated
(Q. to
hESCs.(D) Immunocytochemical analysis cellsat day 10 showinguniform
of coex-
pressionof Pax6 and Rx (merged image includes ToPro-3nuclearstain).(£) FACS
analysisconfirming rapidlossofOct4expression the onsetofbothPax6and
the and
Rxprotein expression.Negative controls FACS
for analyses indicated thewhite
are by
histograms. (Fand G) qPCR (f) and Western (G) the
analysis demonstrating endog-
enous expression the BMP and Wnt antagpnists
of Nogginand Dkk-1.(H) qPCR
Fig. 1. Commitment toward a retinal lineage occurs as a stepwise process,
showingthe nearcompletelossof Pax6and Rxgene expression cellstreatedwith
in
beginningwiththe establishment the eye fieldwithinthe anteriorneuroep-
of
BMP4and Wnt3A.(Scale bar,40 /am.)
ithelium. Each majorstage inretinogenesiscanbe distinguished partbythe
(A) in
expressionof varioustranscription factors.(B) Schematicof the differentiation
protocol used to generate cells of a retinallineage. (Q RT-PCR analysisof the
The onsetof Pax6 and Rx expression detected day6, when
was by
changes in gene expressiontoward an eye fieldfate throughthe first days of
16
approximately of all cellsexpressed
(D-F) Immunocytochemistry typicalhESC aggregates 10 days
differentiation. of 25% thesefactors. Expression
of Pax6 and Rx surpassed
demonstrating expressionof the anteriorneuraltran-
afterdifferentiation, the 90% ofcellsbyday 10 ofdifferentiation
scriptionfactor Otx2 (D), the eye field transcription factor Lhx2 (£), and the to
and increased greater than95% byday 16. Conversely, protein
definitiveneuraltranscriptionfactorSox1 (F). (Scale bar, 200 pirn.)expression Oct4 decreasedto an undetectable
of levelbyday10 of
differentiation. generation a highpercentage cellswith
The of of
eyefield characteristicstheabsenceofexogenous
in Wntand BMP
coloniesexpressed Lhx2(Fig. ID), Otx2(Fig. IE), and Soxl (Fig. antagonists prompted further investigation the endogenous
into
IF) by day 10 of differentiation. expression Dkk-1and Nogginin differentiating cultures.
of hESC
Eyefield cellsareoften characterized thecoexpression Pax6
by of Both geneswere up-regulated during fieldspecification
eye (Fig.
Western de-
the
and Rx (7, 16). Therefore, gene and protein expression ofthese 2F) as determined qPCR. Furthermore,
by analysis
tected proteinexpressionof Dkk-1 and Noggin at day 10 of
twotranscription factors examined further
was in detail.RT-PCR
differentiation Addition Wnt3A BMP4 tocultures
of and
and quantitative PCR (qPCR) analysesrevealed the onset of overthefirst (Fig.2G).differentiation
10 daysof abolished boththeexpres-
expressionof both Pax6 and Rx withinthe firstfew days of sion of Pax6 and Rx (Fig. 2H) and the appearanceof neuroepi-
differentiation 2A-C), which was closelycorrelated withloss thelialcolonies was also
(Fig. (Fig. SI). Endogenous FGF signaling
ofOct4expression. theprotein
On nearly cellscoexpressed involved the acquisition earlyeye fieldfeatures,
level, all in of since the
bothPax6 and Rx within daysof differentiationdetermined addition SU5402, a potentand specific
10 as of inhibitor theFGFR1
of
by immunocytochemistry ID). Cell populations
(Fig. were then receptor, to a complete
led loss ofbothPax6 and Rx expression at
analyzed FACS overthefirst daysofdifferentiation IE).
by 16 (Fig. day 10 of differentiation S2).
(Fig.
Meyeret al. PNAS | September29. 2009 | voi. 106 | no. 39 | 16699

4. of
Acquisition Optic and
Vesicle Optic CupCellPhenotypes. next The
phase in retinal specification vivooccurswiththeformation
in of
theopticvesicles from pairedeye fields. thisstage,all cells
the At
that giveriseto either neuralretina theRPE express
will the or the
transcription factor Mitf of +
(17). The subset Mitf cellsdestined to
becomeneuralretina subsequently down-regulate inresponse
Mitf
to theonsetof ChxlO(also calledVsx2) expression 19). When (18,
eye fieldrosettes were liftedand grownas neurospheres, near
uniform expression Mitfprotein
of was observedwithin14.7 ±
2.1% of all spheresby day 30 of differentiation 14). qPCR
(Fig.
analysisfurther demonstrated that gene expression Mitfin-
of
creasedfrom 16 to day37 ofdifferentiation 3B). Next,
day (Fig. the
relationship betweenMitfand ChxlO protein expression was ex-
amined overtime. ChxlOexpression only was rarely observed day
at
30 (Fig. 3C). Coexpression Mitf
of and ChxlOwas prevalent day by
40 (Fig. 3D), followed mutually
by exclusive expression ChxlO
of
and Mitfbyday50 as Mitfexpression diminished within ChxlO+
neurospheres (Fig. 3E). qPCR analysis that
confirmed ChxlOgene
expression delayedrelative Mitf(Fig. 3F). Similar Mitf,
was to to
ChxlO protein was eventually detectedin nearlyall cells of the
subsetof neurospheres whichitwas expressed
in (Fig. 3G). Quan-
tification ChxlO proteinexpression
of demonstrated that18.0 ±
3.3% of all neurospheres containedChxl0+ cells by day 40 of
differentiation (Fig. S14), and withinthese ChxlO-expressing
spheres, 90.7% ± 5.2% of cells expressed ChxlOby day 50 (Fig.
S3B). ByFACS, 26% oftheentire culture cell population expressed
ChxlOat day40 (Fig. 3H). The remaining ChxlO-negative neuro-
spheres derived from early field population
the eye cell maintained
a neuralfateas indicated expression Soxl and ßlll-tubulin
by of
(Fig. S4 A-F, and J). Non-retinal neurospheres also expressed
forebrain markers, including Otx2(Fig. S4 G-I, and/), butdid not
express endoderm (alpha-fetoprotein), mesoderm(brachyury),
hindbrain (HoxB4), or midbrain (En-1) markers (Fig. S4/).
Amongthoseindividual cellsthatexpressed ChxlO, greater than
99% maintained expression Pax6,which a requirement early
of is of
retinal progenitor cells (20) (Fig. 31). Furthermore, manyof the
+
ChxlO clusters were arrangedin rosetteswith cells oriented
radially awayfroma core thatwas positive the tight for junction
protein ZO-1 (Fig. 37), a feature associatedwithprogenitor pop-
ulations (14). Whileclusters contained
that Chxl0+ cellsincluded
a smallnumber ßlll tubulin+neurons
of (Fig.3K),thesecellsrarely
coexpressed ChxlO.Thus,ChxlOexpression associatedwas witha
neural cell type that had not yet acquired a matureneuronal
phenotype. Fig. 3. Acquisition of optic vesicle and optic cup cell phenotypes. {A) Mitf
Giventhepotential roleof FGF signaling thespecification
in of protein expression in neurospheres after30 days of differentiation. qPCR
(B)
theneuralretina(21), we nextexamined effect thespecific
the of analysisof Mitfgene expressionover the first days of differentiation.
80 (C-£)
The Immunocytochemical analyses of the time course of Mitfand ChxlO protein
FGF inhibitor SU5402, on Mitfand ChxlOgene expression.
additionof SU5402 to adherenthESC cultures the optic expression in neurospheres at 30 (Q, 40 (D), or 50 (£) days of differentiation.
during (F) qPCR analysis of ChxlO gene expression over the first days of differen-
80
vesicleand opticcup stagesofdifferentiation 16-40) resulted
(days tiation. (G) Uniform Chx10 expressionthroughouta subset of neurospheresby
in an 11.8-fold increasein Mitfgene expression day 40, as at day 40. (H) FACSanalysisdemonstratingthe percentage of all cells expressing
measuredby qPCR (Fig. 3L). By contrast, ChxlO expression was ChxlO at day 40. (I) Immunocytochemicalanalysis showed all Chx10+ cells
reduced15.9-fold a result thistreatment.
as of coexpressed Pax6 at day 40. (J) Rosettes of Chx10+ cells expressed the tight
junction proteinZO-1 withintheircore. (K) Rare Chx10+ cells coexpressed j3lll
DifferentiationRetinal Types
of Cell from hESC-Derived Retinal Pro- tubulin at day 40. (/.) qPCR demonstrating increased Mitf expression and
genitors.The RPE is the first differentiatedretinalcell typeto correspondingdecreased ChxlO expression in adherent culturestreated with
+ the FGFinhibitor SU5402. qPCR values were expressed as fold change relative
appear during retinogenesis,arisingfroma populationof Mitf
(Scale bars, 500
to culturesat day 16 (B and F) or day 10 (L) of differentiation.
andPax6+ cellspresent theouterlayer theearly
in of opticcup (21). /mm panels A and G; 50 /¿m panels C-E,J,and K; and 75 /xm panel /;blue
in in in
WhenPax6+/Rx+ eyefieldrosettes weremaintained adherent
as stain in A and G is Hoechst nuclear dye.)
cultures,distinct of
patches polygonal, pigmented wereinitially
cells
observed approximately 30 ofhESC differentiation 44).
at day (Fig.
These cellsmaintained expression thetranscription
of factor Mitf,
while also expressing RPE-associatedtight
the junctionprotein Prolongedmaintenance the hESC-derivedretinalprogen-
of
ZO-1 (Fig.AB).Atday40 ofdifferentiation, analysis
FACS revealed itorsas neurospheresallowed for further maturation these
of
that25% of all adherent cellsexpressed Mitf,and 77% of all cells cells towarda photoreceptor phenotype.Among the first dif-
expressed Pax6 (Fig. AC). RT-PCR analysis demonstrated main- ferentiatedneuralretinalphenotypes observedduring develop-
tainedexpression Pax6 in thiscell population
of overtime, well
as mentare cone photoreceptors 23), whoseprecursors
(22, express
as theacquisition moremature
of RPE-associated markers suchas the primitivecone and rod photoreceptor-specifictranscription
RPE65 and bestrophin (Fig. AD). factorCrx (24). By day 80 of differentiation, ± 3.1% of all
19.4
16700 | www.pnas.org/cgi/doi/10.1073/pnas.0905245106 Meyeret al.

5. >-
oc
<
h-
z
LU
cell lineingreater the
detail.Upon differentiation, appearances of O
'J
the iPS cell colonies, iPS cell aggregates, neural rosettesand m
ut
t/i
neurospheres indistinguishable those hESCs (Fig.S7).
were from of
Duringdifferentiation, immunocytochemistry revealedearlyeye
field cellscoexpressing Pax6and Rxbyday10 (Fig.6A). Thesecells
also expressed fullcomplement eye fieldand neuroepithelial
a of
transcription factors (Fig. S8). Discretepopulations Mitf cells
of 4-
wereobserved uponfurther differentiationeyefield
of coloniesas
neurospheres (Fig. 6B). Like theirhESC counterparts, manyof
these iPS cell neurospheres appeared to lose Mitfexpression in
favorof ChxlO expression (Fig. 6C), yielding neurospheres that
werehighly enriched Chxl0+ cells(Fig. 6D). Amongthetotal
for
population, 12.9 ± 4.3% ofall neurospheres expressed ChxlOat 40
days of differentiation, withinwhich90.1 ± 1.2% of all cells
expressedChxlO. Over time,photoreceptor markers appeared,
such as the rod-and cone-specific transcription factor Crx,which
was present 14.4 ± 5.1% ofall neurospheres day80 (Fig. 6E).
in by
Similar the expression earliermarkers retinal
to of of differentia-
tion,Crx+ cells were commonwithin individual positive neuro-
spheres, constituting ± 9.3% of cells.At day80,44.6 ± 8.1%
65.5
of cellswithin Crx+ clusters expressed recoverin (Fig. 6F) and/or
opsin (Fig. 6 G and H). As with hESCs, recoverin and opsin
expression was not found in Crx-negative cells. PCR analysis
Fig. 4. Generation of retinal pigment epithelium. (A) Photomicrographof confirmed sequence and timing gene expression these
the of of
adherent culturesshowing pigmented, hexagonal RPE-like cells. (6) Immuno- markers, alongwith early ofOct4expression
the loss (Fig.61).RPE
staining revealing expression of MitfwithinRPE-like cells, as well as the tight cellswerepresent within cellcultures well,
iPS as with pigmentation
junction protein ZO-1. (Q FACS analysis demonstratingthe percentage of all first apparent approximately 35 ofdifferentiation typical ¿S
at day and
adherent cells expressing Mitf and Pax6 at day 40 of differentiation.(D)
RT-PCR studies showing expressionof genes associated withan RPEfate. (Scale
monolayers arising day 50 (Fig. 67). Like hESC-derived
by RPE,
thesecellspossessedmorphological characteristics mature
of RPE
bars, 100 /im.)
and expressed Mitfand ZO-1 (Fig. 6K).
Discussion
neurospherescontained Crx+ photoreceptor precursors(Fig.
The results
63.0 ± 7.6% ofall cellsexpressed presented heredemonstrate thathumanpluripotent
5^4).Within theseneuropheres,
Crx. Furthermore, 46.4 ± 7.9% of Crx+ cells expressedmore stemcells can adopt signature features associatedwithall major
mature photoreceptormarkers,such as recoverin (Fig. 5B) stages of earlyeye and retinaldevelopment, whilefollowing an
and/or cone photoreceptor-specific
the expected timeline for human retinal development(22, 23).
proteinopsin (Fig. 5C).
Recoverin and opsin expression was not observed in Crx- Althoughpreviousreports have shownthathESCs can acquire
retinal characteristics various times duringdifferentiation
at
negativecells. used to identify retinalcell types
To analyze the time course and sequential acquisition of (3-7), manymarkers primitive
are also expressedin otherdevelopingneuralcells. This makes
neuroretinal-and photoreceptor-associated gene expression,it difficult unequivocallyassign immature
to cell typesto the
RT-PCR analysis was performed(Fig. 5D). Throughoutthe
retinallineagewithout knowledgeof theirdynamic behaviorin
differentiation process fromday 16 through day 80, Pax6 geneculture.Thus, it is important monitor
to each stage of cellular
expressionwas detected. Rx gene expressionwas also present maturation ensurethatcritical
to checkpoints are
developmental
earlyin differentiation, followedby the consecutiveexpression
met in order and withina predictabletimeframe.
of ChxlO,Crx,and opsin.Overall,thetiming expression the
of of In the first weeksof humandevelopment, portion the
few a of
gene and proteinmarkers used in thisstudy coincidedwiththat
primitive anterior neuroepithelium risetotheeyefield
gives (13, 16,
of normalhumanretinaldevelopment(22, 23) (Fig. S5). of
26, 27), a cell populationcharacterized the expression
by
During normal retinogenesis, Pax6(+5a) isoformis ex-
the numerous transcription factors including Pax6,Rx, Six3,Six6,Til,
pressedin increasing abundancerelative total Pax6 (25). RT-
to and Lhx2.We have demonstrated the production Pax6+/
that of
PCR results fromthe presentstudysimilarly suggested thatthe
Rx+ cellsis highly efficient, 95% ofall cellscoexpressing
with these
Pax6(-l-5a) isoform becamemoreprevalent during hESC differen-
essential factors. efficiencylikely inpartto
This is due
transcription
tiation(Figs.2A and 5D). To verify observation,
this qPCR of thea relativelack of influencefromendogenousBMP and Wnt
Pax6(+5a) isoform relative totalPax6expression performed
to was
signaling, since both pathways knownto antagonizeneural
are
(Fig. S6). This analysis confirmed onsetof Pax6(+5a) expres-
the
specification (28, 29). In support thistheory,
of increasing expres-
sionbetweendays4 and 16 of differentiation demonstrated
and a
sionofBMP andWntantagonists and
(Noggin Dkk-1, respectively)
relative in
increase theexpression thisisoform
of betweendays60 was observedin hESC cultures shortly after onsetof differen-
the
and 70, whichcorresponded the appearanceof photoreceptor-
to tiation.Early exposureof differentiating hESCs to recombinant
likecells in culture. BMP4 andWnt3aeliminated expression Pax6and Rx,as well
the of
as the subsequent formation neuralrosettes.
of
Differentiation of Retinal Cell Types from Human iPS Cells. To AlthoughPax6 and Rx have been used to identify retinal
determine potential stepwise
the for derivation retinal types progenitor
of cell cells in differentiating cultures(7, 16), during
ESC
from human cells, appliedthehESC differentiation
iPS we protocol development are
thesefactors intially coexpressed a broadregion
in
to fourdifferent humaniPS cell lines.Consistent witha previous of the anterior neuralplate thatincludes eye fieldand future
the
report (11), considerable variation found theability these forebrain
was in of (16). Thereafter, Pax6+/Rx+ cellsbecome restricted to
linesto producePax6+ neuroectodermal at day 10 of differ- morespecific
cells areasofthedeveloping CNS (16), predominantly the
entiation, efficiencies
with rangingfrom to 56% ofthetotalcell retina(26, 30). In the presentstudy,
5% the majority the early
of
population. Based on theseresults, choseto study IMR90-4
we the Pax6+/Rx+ populationdid not subsequently adopt cellularphe-
Meyeret al. PNAS | September29, 2009 | vol.106 | no. 39 | 16701

6. Fig. 5. Generationof earlyphotoreceptorphenotypes.(A) Immunocytochem-
¡cal detection of cells expressing the photoreceptor-specif transcription
ic factor
Crx at 80 days of differentiation. and O Expressionof the photoreceptor
(B
protein recoverin(B) and the cone photoreceptor-specific protein opsin (O
among Crx-expressing cells at day 80. (D) RT-PCRdemonstratedthe stepwise
acquisitionofa cone photoreceptor fatefrom eye fieldpopulation.(Scale bars,
an
50 /xm.) Blue stain in A is Hoechst nucleardye.
notypes theoptic
of vesicle opticcupdespite
or retaining anterior
an
neuralidentity. Therefore, enriched
the Pax6+/Rx+ cell popula-
in
tionderived thisstudy mostcloselyresembled primitive
a stage
ofhuman field
eye development, which the
preceded appearance of
committed retinal progenitors.
Afterthe optic vesiclesevaginatefromthe paired eye fields,
expression Mitfoccursthroughout fatedto become retina
of cells
(12, 17). However,the decisionto differentiate towardeithera
neuralretina RPE fateis revealedduring late opticvesicle
or the
and optic cup stages,in part via differential expression the
of
transcription factorChxlO (18, 19). Neural retinalprogenitors
destinedforthe innerlayerof the opticcup expressChxlO and
down-regulate in response FGFs secreted theoverlying Fig.6. Stepwise retinalspecificationfromhuman ¡PScells. {A) Variousstages
Mitf to by
surfaceectoderm. Thus, ChxlO is the earliestspecific marker of of retinaldifferentiationwere observed, beginningwith Pax6+/Rx+ eye field
neuralretinal progenitor cells (19). Conversely, cells destined for cells by day 10. (B-D) Mitf+ and Chx10+ cells, indicativeof the optic vesicle/
theouterlayer theopticcup remain
of +
Mitf and ChxlO-negative optic cup stages, are evident by day 40. (£) By day 80, clusterswere present
and subsequently differentiate into RPE. Our resultsprovide containing Chx10+ retinal progenitors and Crx+ photoreceptor precursor
cells expressed the photoreceptor protein
evidencethathESCs proceed through analogousstagesof early cells. {F-H) Many Crx-expressing
recoverin(F) and the cone-specificproteinopsin (G and H). (/)RT-PCR analysis
retinaldifferentiation,indicated the spatiotemporal
as by expres-
sionofMitf ChxlOinneurospheres.
and inhibition demonstrating the stepwise expression of retina- and photoreceptor-
of associated
Furthermore, genes in differentiating ¡PS cells over time. (J and K) RPE cells
endogenous FGF signaling during opticvesicleand opticcup derived fromiPS cells acquired a typicalhexagonal morphologyand pigmen-
the
stagesof hESC differentiation in
resulted a profound increasein tation (J)and expressed Mitfand ZO-1 {K). (Scale bars, 50 /am.)Blue stain in B
Mitf geneexpression a corresponding
and decreasein ChxlOgene and D is Hoechst nuclear dye; blue stain in F is To-Pro-3nuclear dye.
expression. This suggeststhat mechanisms governing cell fate
choicein thedeveloping retina mayalso function differentiating
in
hESC cultures. differentiation. observationis consistent
This withnormalret-
After adopting a retinalfate, individual neurospheres yielded a inal development,where early cell types often give rise to
high percentageof photoreceptor precursors a time frame multipledistinct
in progeny the same lineage. However,there
of
predicted by normal humanretinogenesis. withAs earlierstages of now exist opportunitiesto introduceexogenous factorsfor
retinaldifferentiation, was achievedwithout additionof definedtimeperiodsto augment
this the production retinal types
of cell
specificexogenous agents.Previously, retinoic and taurine
acid had at specificdevelopmental stages. Such precisionis likelyto be
been used to inducedifferentiationphotoreceptor-like (7).
of cells important, since a single factorcan have diverse effectson
By eliminating such agents,the presentsystem suitedforthe cellularfatechoice depending thestageofdevelopment
is on (31).
investigation of endogenousfactors and mechanisms thataffect For example,we observed thatearlyinhibition endogenous of
differentiation maturation specific
and of retinal types.
cell in
FGF signaling differentiating hESCs resulted a loss of eye
in
Taking intoaccounttheentire hESC populationpresent the field specification,
at whereas later inhibition differentially regu-
startof the differentiation process,we observed a decrease in latedgenesimportant theinduction RPE and neuralretina
for of
targetedcell production witheach subsequentstage of retinal progenitors.Manipulation of the culture environment with
16702 | www.pnas.org/cgi/doi/10.1073/pnas.0905245106 Meyer al.
et

7. |
signaling factors may also alter the time course of retinalcell In summary, haveshownthathESCs meetthecriteria 2)
we (1,
differentiation fromhESCs. This is suggestedby the striking to serve as a comprehensive vitromodel system human
in for
in
differences the timing photoreceptor
of markerexpression retinogenesis. Usingan identical culture method, humaniPS cells
observedin previousstudies,in whichthe onset of Crx expres- showa similar potential, although variation occurbetween
can lines.
sion rangedfromone to thirteen weeks (6, 7). On a broaderlevel,thisstudy a
supports roleforpluripotent stem
Giventheability hESCs to mimic
of normalhumanretinogen- cells to testconceptsin humandevelopmental biologythatwere
sourceofhumanpluripotent previously extrapolated fromanimalmodels.In turn, thisability
esis,we investigated whether another
stem similar A previous could narrow gap between understanding humandevel-
the our of
cells,iPS cells, displayed a potential. report
opmentand thatof othermammalian species.
byYu et al. (11) showedthathumaniPS cell linesdiffered their in
earlyexpression Pax6, a finding
of confirmed here. Since Pax6 Methods
expression necessary retinal
is for development, is notsurprising Maintenance of hESCs and Human iPS Cells. Pluripotent stem cells were
it
thatone of the highest Pax6-expressing lines fromthat study, maintained as previouslydescribed for hESCs (15). Detailed protocols are
IMR90-4, wasefficient producing
at retinal populations.
cell Other available in the 5/Text
iPS celllinesdisplayed reduced competency to produceneuraland
retinal types, phenomenon observed Hirami al. (8).
cell a also by et Differentiation hESCs and Human iPS Cells. The initial differentiationof
of
Therefore, present techniques forderiving cells from
iPS somatic hESCs and human iPS cells toward an eye field fate was performed with
modificationsto previouslydescribed protocols (15, 36). Thereafter,a chem-
cells do not always yielduniform lineagecompetencies between
ically-defined retinaldifferentiation medium was used to promote the step-
lines. wise production of retina-specific types fromfree-floatingneurospheres
cell
A detailedknowledge the stagesand timecourseof retinal (36). Detailed protocols are available in the 5/Text
of
differentiation hESCs and iPS cells not only providesan
from
opportunity studyfundamental
to questionsof human retinal RT-PCR.RT-PCRand qPCR experiments were performed as previouslyde-
development, mayalso aid efforts use pluripotent
but to stemcell scribed (36). More detailed methods, including primer sequences, can be
found in the SI Text as well as Table S1.
for
derivatives pharmaceutical testing and retinalrepopulation
A
studies. previous report MacLaren et al. (32) demonstrated Immunocytochemistry. aggregates were plated onto coversiips,fixedwith
by Cell
that a
cellsfrom specific stageof mouseretinal development were 4% paraformaldehyde,and then immunostained as described (15). Detailed
capable of functionally integrating degenerateadult mouse procedures are provided in the SI Textas well as Table S2.
into J 5
retinas. Morerecently, Lamba et al. (33) notedsimilar resultsusing
a mixture retinal
of cell typesderivedfromhESCs. The hESC FACS. Staining and sorting of cells were performed as previouslydescribed
Detailed procedures are provided in the 5/Text
differentiation protocoldescribedin the presentstudyprovides (15).
access to humanretinal cells at all majorstagesof retinal devel-
opment. The nearabsenceof contamination from non-neural cell WesternAnalysis.Western blots were 5/Textas well previouslydescribed (36).
Detailed procedures are provided in
performedas
as Table S2.
types and the potentialto enrichfor discreteretinalcell types
further to the possibleclinicalutility thesedifferentiatingACKNOWLEDGMENTS. We thank B. Hu, T. Lavaute, and M. Pankratz for
add of
cultures. potential iPS cellsto generate
The for multiple retinalcell technical assistance,and the staffat WiCell forpreparation of MEF. Thiswork
types willaid inthedevelopment invitroof models human
of retinal was supported by National Institutes of Health Grants K08EY015138 (to
D.M.G.) and R01NS045926 (to S.C.Z.), the Foundation Fighting Blindness
degenerative diseasesand stimulate investigation customized (D.M.G.), the Walsh Consortium(D.M.G.), the LincyFoundation (D.M.G.), and
into
cell for
stem therapies patients afflicted thesedisorders 35).
by (34, a Retina Research Foundation Gamewell Professorship D.M.G.)
(to
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