STEM CELLS AND DEVELOPMENT
Volume 19, Number 10, 2010
© Mary Ann Liebert, Inc.
Subfractionation of Differentiating Human Embryonic Stem
Cell Populations Allows the Isolation of a Mesodermal
Population Enriched for Intermediate Mesoderm
and Putative Renal Progenitors
S. Adelia Lin , 1–3 Gabriel Kolle , 4 Sean M. Grimmond , 4 Qi Zhou , 2,3 Elizabeth Doust , 2,3 Melissa H. Little , 4
Bruce Aronow , 5 Sharon D. Ricardo , 6 Martin F. Pera , 7 John F. Bertram , 1 and Andrew L. Laslett 1–3
1 Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia.
2 Australian Stem Cell Centre, Clayton, Victoria, Australia.
3 Molecular and Health Technologies, CSIRO, Clayton, Victoria, Australia.
4 Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia.
5 Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio.
6 Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, Clayton, Victoria, Australia.
7 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern
California, Los Angeles, California.
Human embryonic stem (ES) cells are pluripotent and are believed to be able to generate all cell types in the
body. As such, they have potential applications in regenerative therapy for kidney disease. However, before this
can be achieved, a protocol to differentiate human ES cells to mesodermal renal progenitor lineages is required.
Reduction of serum concentration and feeder layer density reduction cultures were used to differentiate human
ES cells for 14 days. Differentiated ES cells were then fractionated by fl ow cytometry based on expression of the
markers CD24, podocalyxin, and GCTM2 to isolate putative renal cells. These cells up-regulated the expression
of the renal transcription factors PAX2, LHX1, and WT1 when compared with unfractionated human ES cells.
Immunohistochemical assays confi rmed that a subset of cells within this fraction co-expressed nuclear WT1
and PAX2 proteins. Transcriptome profi ling also showed that the most differentially up-regulated genes in this
fraction preferentially associated with kidney development in comparison with any other lineage. When com-
pared with a transcriptome profi le database of urogenital development (GUDMAP), the top 200 differentially
up-regulated genes in this fraction strongly clustered into a group of genes associated with the metanephric
mesenchyme at E11.5 and the corticonephrogenic interstitium at E15.5 of murine kidney development. Hence,
this approach confi rms an ability to direct human ES cells toward a renal progenitor state.
exhaustible source of cells for the study of physiological
diseases and are an excellent model system for the study
of development. However, it is arguably their potential as
a renewable source of specialized cells for cell-based thera-
pies that has stimulated much research in the ES cell fi eld.
Indeed, human ES cells have been successfully directed to
differentiate in vitro using a variety of methods into many
cell types including neural progenitors and their differen-
tiated progeny [ 1 , 2 ], endothelial cells [ 3 , 4 ], osteogenic cells
[ 5–7 ], cardiomyocytes [ 8 , 9 ], insulin-producing cells [ 10 , 11 ],
hepatocytes [ 12 , 13 ], keratinocytes [ 14 , 15 ], germ cells [ 16 , 17 ],
and trophoblast cells [ 18 , 19 ]. However, differentiation to the
mbryonic stem (ES) cells are pluripotent and have un-
limited self-renewal properties. They represent an in-
renal lineage has not yet been examined in detail. Murine ES
cells have been shown to be able to integrate with E12–E13
metanephros [ 20 ] and have also been demonstrated to have
the capacity to differentiate into renal epithelial cells that
can integrate into developing kidneys [ 21 , 22 ]. In the case of
human ES cells, kidney-like structures have been observed
in teratomas formed from human ES cells [ 23–25 ] and reverse
transcription-polymerase chain reaction (RT-PCR) analyses
of heterogeneous human ES cell-derived populations in
embryoid bodies have indicated the presence of transcripts
associated with kidney development [ 26 ]. In addition, in a
recent report by Batchelder and colleagues, genes associated
with the intermediate mesoderm and developing kidney
were shown to be expressed in human ES cell colonies cul-
tured in the presence of retinoic acid, activin A, and BMP7
LIN ET AL. 1638
Histological sections of fi rst trimester human embryos
were obtained from archival material donated with informed
consent by patients undergoing termination of pregnancy
at the John Radcliffe Hospital, Oxford, UK. The tissue was
fi xed in absolute alcohol, embedded in paraffi n, and sec-
tioned at 5-μm thickness. All tissue was obtained following
local ethical committee approval.
Teratomas were formed as described previously [ 38 ]. In
brief, ~50,000 human ES cells were injected beneath the testis
capsule of SCID mice between 5 and 6 weeks of age. The ani-
mals were monitored weekly beginning at around 4 weeks
for tumor development. Lesions usually became apparent
as swellings in about 5 weeks and teratomas removed at ~6
weeks. The tumors were removed, fi xed in formalin, and
sent for routine histological processing. Every 10th section
was visually examined for kidney-like areas after hematox-
ylin and eosin staining.
was performed on the human embryo and teratoma sec-
tions using the Vectastain ABC kit (Vector Laboratories,
Burlingame, CA) as per the manufacturer’s instructions.
Embryo sections were subjected to antigen retrieval in 10
mM citric acid buffer (pH 6.4) prior to immunohistochem-
istry. Primary antibodies used were mouse anti-cadherin
16 and mouse anti-cadherin 11 (Zymed Laboratories, San
Francisco, CA), mouse anti-human WT1 (Dako, Carpinteria,
CA), mouse anti-podocalyxin (PHM5, kind gift from Dr.
David Nikolic-Paterson, Department of Nephrology, Monash
Medical Centre), and mouse anti-CD24 (BD Pharmingen, San
Diego, CA). Isotype-matched negative control antibodies
(Dako, CA) were also employed to ensure specifi c staining.
Flow cytometric analyses of differentiated
human ES cells
For fl ow cytometric analyses with CD24 (BD Pharmingen,
CA), podocalyxin, and GCTM2, colonies were incubated
with Dispase (Gibco, Carlsbad, CA) at a concentration of 10
mg/mL in human ES media [ 24 ], lifted intact by nudging
gently with a 1,000-μL pipette tip and transferred to a 15-mL
tube containing 5 mL phosphate-buffered saline (PBS). Cells
were then dissociated into single cells by incubation in
TrypLE™ Express (Gibco, Carlsbad, CA) at 37°C for 3 min
after which the reaction was inactivated by the addition of
an equal volume of serum containing human ES cell media.
Harvested cells were blocked in 3 mL 1% normal goat serum
in PBS and stained with CD24, podocalyxin, and GCTM2
antibodies diluted in 1% normal goat serum in PBS. A nega-
tive control tube was included comprising cells labeled with
secondary antibody only. Just prior to sorting, cells were
transferred to a 5-mL polypropylene tube and the viability
dye propidium iodide (Sigma, St. Louis, MO) was added to
the cells at a concentration of 1 μg/mL to distinguish between
live and dead cells using the BD FACSVantage DiVA cell
sorter (Becton Dickinson, Franklin Lakes, NJ). Sorting was
on different substrates [ 27 ]. We sought to provide further
evidence that human ES cells have the capacity to differen-
tiate along this lineage so as to assess the potential utility of
human ES cells in renal research and regenerative medicine.
The permanent postnatal kidney, the metanephros, is de-
rived from 2 intermediate mesoderm-derived structures: the
ureteric bud (UB) and the metanephric mesenchyme (MM)
[ 28 , 29 ]. While the UB is a critical inducer tissue for the forma-
tion and patterning of the functional units of the kidney, the
nephrons, this tissue ultimately only gives rise to the calyceal
system of the kidney, collecting ducts, and the ureter. It is
the MM that is regarded as the renal progenitor population
as this tissue gives rise to all remaining segments of the
nephrons including the glomeruli, as well as contributing ex-
tensively to the interstitial elements of the kidney, including
portions of the vasculature. For this reason, the generation
and competent culture of MM is a major target for renal re-
generation and bioengineering. The paradigm for directed
differentiation of human ES cells suggests that the cells
will need to recapitulate the normal steps of embryology to
generate a specifi c cell type. However, as differentiation of
human ES cells occurs in the absence of the intrinsic struc-
tural architecture present in a developing embryo, combina-
tions of specifi c antigenic and molecular markers identifi ed
from mammalian embryological studies are required to cat-
egorically identify the target cell population within the mix-
ture of other cell types also present in cells differentiating
from human ES cells. In the case of kidney differentiation,
specifi c markers of MM are required. Microarray investiga-
tions coupled with in situ hybridization analyses conducted
by Challen and colleagues [ 30 ] revealed that the cell surface
marker CD24 was present in the mouse MM from E10.5 while
podocalyxin is expressed in both MM and the surrounding
intermediate mesoderm. Gene-targeting studies have identi-
fi ed the transcription factors Pax2 , Lim1 (LHX1) , and Wt1 as
critically important for kidney development [ 31–33 ]. In this
study, to assess potential renal differentiation we employed
a differentiation regime of low serum concentration that has
previously been shown to permit cardiomyocyte and defi n-
itive endoderm differentiation [ 34–36 ], in combination with
a reduction in the density of the mouse embryonic fi broblast
(MEF) feeder layer to induce human ES cell differentiation.
Coupled with this differentiation regime, we have utilized
combinations of the markers listed above to prospectively iso-
late cell populations displaying a similar phenotype to MM
from a heterogeneous population of differentiated human
ES cells. Quantitative PCR, immunofl uorescence staining,
and gene microarray analyses are also presented to demon-
strate the identifi cation of a specifi c fraction enriched for cells
expressing genes associated with the developing kidney.
Materials and Methods
Human ES cell culture and differentiation
Human ES cell lines HES-2, HES-3, and HES-4 [ 37 ] were
maintained as previously described [ 24 ] in the presence of
20% fetal calf serum on a layer of MEFs at a density of 6 × 10 4
cells/cm 2 . For differentiation experiments, human ES cells
were cultured on a reduced density (2 × 10 4 cells/cm 2 ) of
feeder cells in 20% serum concentration for 2 days and 5%
serum concentration for 12 more days with a media change
every second day.
DIFFERENTIATION OF HES CELLS TO PUTATIVE RENAL PROGENITORS
RNA samples were analyzed for integrity using the Agilent
Bioanalyzer 2100 and an RNA Integrity Number (RIN) was
obtained using the Agilent Expert 2100 software vB.02.02.
Samples with a RIN score of above 8.5 were deemed suitable
for array analyses. Subsequently, 200 ng of total RNA from
each sample was amplifi ed using the Illumina ® TotalPrep™
RNA Amplifi cation Kit (Ambion, Foster City, CA) accord-
ing to the manufacturer’s guidelines with a 14-h in vitro
transcription step. The 1.5 μg of amplifi ed cRNA per array
was hybridized to Human-6 v2 BeadChip arrays accord-
ing to the manufacturer’s instructions and detected using
Fluorolink Streptavidin-Cy3 (GE Healthcare Biosciences,
Freiburg, Germany). BeadChip arrays were scanned using
the BeadStation array scanner (Illumina, San Diego, CA) and
raw image intensity values were compiled using the Illumina
BeadStudio software v2.3.41. The raw array data was nor-
malized in R , a language and environment for statistical
computing and graphics (www.r-project.org) before being
imported into GeneSpring GX v7.3.1 (Agilent Technologies,
Santa Clara, CA) for visualization.
Cytospinning FACS-fractionated cells
and immunofl uorescent labeling
Fluorescence-activated cell sorter (FACS)-sorted cells were
washed twice in PBS + and transferred in a small volume of
PBS + to V-bottomed tubes containing 3 mL of 4% paraformal-
dehyde and incubated for 10 min at room temperature. Cells
were then washed and cytospun onto poly- l -lysine-coated
slides in the Shandon Cytospin ® 4 cytocentrifuge at 7 g for 10
min at low acceleration. Cytospun slides were then washed
in a Coplin jar containing Milli-Q ® H 2 O with shaking for 5
min before permeabilization with 0.1% Triton X-100 in PBS for
10 min at room temperature. Slides were then marked with a
wax pen and blocked with 10% normal goat serum in PBS + at
room temperature for 30 min. WT1 (Dako, Carpinteria, CA)
and PAX2 (Zymed Laboratories, San Francisco, CA) antibod-
ies diluted with blocking buffer were added to the slides and
incubated for 1 h at room temperature or at 4°C overnight.
performed with a nozzle size of 80 μm and a sheath pressure
of 30 psi into 5-mL collecting tubes containing 1 mL human
ES cell media. Cells were initially gated based on forward
and side scatter to exclude cellular debris and nonviable
cells were excluded by their uptake of the viability marker,
propidium iodide. Subsequently, gates for each marker were
set using negative controls.
Total RNA isolation and quantitative PCR
Total RNA was isolated using the RNAqueous ® -Micro
RNA Isolation Kit (Ambion, Foster City, CA) and reverse-
transcribed using Superscript™ III Reverse Transcriptase
(Invitrogen, Carlsbad, CA) according to manufacturer’s
instructions. Real-time quantitative gene expression analy-
ses were performed using the Taqman ® gene expression
assays (Applied Biosystems, Foster City, CA) using prede-
signed Taqman ® probes for the genes of interest ordered
from the manufacturer according to supplied instructions
( Table 1 ). For each reaction, 0.5 μL of cDNA template was
incubated with 5 μL Universal Master Mix, 0.5 μL 20× pre-
designed probe, and 4 μL water in one well of a MicroAmp™
Optical 96-well Reaction Plate (Applied Biosystems). The
thermal cycling conditions were as follows: 50°C for 2 min,
95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C
for 1 min. Gene expression levels were normalized to the
housekeeping gene 18S rRNA and transcript abundance of
each fraction was compared with those in the unfraction-
ated global population. For validation of microarrays, gene
expression levels normalized to the housekeeping gene 18S
rRNA were calibrated against a large stock of cDNA of an
in-house undifferentiated human ES cell line [ 39 ].
RNA amplifi cation and microarray analyses
Total RNA preparations (4 biological replicates of sepa-
rate passage numbers for HES-4) with a minimum concen-
tration of 20 ng/μL and an A260/A280 ratio of >1.8 were
sent to the SRC Microarray facility at the University of
Queensland for RNA amplifi cation and microarray analyses.
Table 1. List of Predesigned Taqman® Probes for QPCR (Applied Biosystems)
Homo sapiens POU domain, class 5, transcription factor 1 transcript variant 1, mRNA
Homo sapiens growth differentiation factor 3, mRNA
Homo sapiens LIM homeobox 1, mRNA
Homo sapiens paired box 2, mRNA
Homo sapiens Wilms’ tumor 1, mRNA
Homo sapiens odd skipped related 1 (Drosophila), mRNA
Homo sapiens mesenchyme homeobox 1, mRNA
Homo sapiens twist homolog 1, mRNA
Homo sapiens CD34 molecule, mRNA
Homo sapiens GATA-binding protein 4, mRNA
Homo sapiens IKAROS family zinc fi nger 1 (Ikaros), mRNA
Homo sapiens forkhead box F1, mRNA
Homo sapiens cadherin 5, type 2, (vascular endothelium), mRNA
Homo sapiens follistatin, mRNA
Homo sapiens T-box 6, mRNA
Homo sapiens transcription factor 15 (basic helix-loop-helix), mRNA
LIN ET AL. 1640
markers indicative of cell types of the kidney. To this aim,
we initially carried out immunological studies in fi rst tri-
mester human embryo sections. Immunoreactivity for WT1,
cadherin 16, and podocalyxin was observed in derivatives
of both the UB and the MM ( Fig. 1A–1F ) although of these
WT1 and podocalyxin were also found in other tissues
both in the developing embryo and the adult. Cadherin 16
expression, although specifi c to the adult kidney, has been
reported in the mouse sex duct and lung [ 40 ]. However, we
did not observe any immunoreactivity to cadherin 16 in
human embryonic sex ducts or lungs, suggesting that in the
human, cadherin 16 expression may be restricted to cells of
These markers were then used to investigate the ability
of human ES cells to differentiate to cells of the nephric lin-
eage in teratomas following ES cell injection into the testis
capsule of SCID mice. Teratomas derived from human ES
cells typically contain a variety of tissues such as muscle,
neural tissue, and various forms of epithelia, with some
showing a high degree of tissue-specifi c organization
[ 23 , 24 ]. However, such structures are usually immature
and therefore defi nitively identifying the tissues present
in such lesions can be diffi cult. In order to identify areas
of potential renal tissue, teratomas were examined micro-
scopically for structures indicative of the kidney. After
Following 2 washes in PBS + , isotype-matched secondary
antibodies (Invitrogen, Carlsbad, CA) diluted in blocking buf-
fer were added and allowed to incubate for 30 min at room
temperature. Slides were protected from the light from this
step onward to prevent photobleaching. After incubation, fi ve
2-min washes were performed as above and cells were labeled
with DAPI (Invitrogen, CA) diluted in PBS + and mounted in
Vectashield ® (Vector Laboratories, Burlingame, CA).
Statistical analyses were performed using the SigmaStat
3.1 Software. The differences between 2 means were non-
parametrically analyzed using the Mann–Whitney rank
sum test. Values were considered to be signifi cantly differ-
ent when the achieved signifi cance level ( P value) was ≤0.05
(denoted by *).
Human ES cell-derived renal structures form
Identifying renal cells from a mixture of other cell types
in a human ES cell-derived teratoma requires the use of
FIG. 1. Immunohistochemical analyses of human embryo sections (A–F) and kidney-like structures within teratomas
(G–J) formed from hESC injected into SCID mice. a. Staining of 8-week embryonic kidney for WT1 revealed cytoplasmic
staining of podocytes (arrow). (B) Isotype-matched negative control for A. (C and D) Staining of 7-week embryonic kidney
with cadherin 16 showed immunoreactivity within nephric duct (black arrow) and MM-derived (white arrow) structures.
(E) Isotype-matched control for C and D. (F) Podocalyxin was localized to nephric duct (black arrow) and MM-derived
(white arrow) structures within the 9.5-week human embryonic kidney. (G) Cadherin 16 staining of a duct-like structure
within a teratoma. (H) WT1 immunoreactivity on rare cells within a teratoma. (I) Podocalyxin staining of a glomerular-like
structure found in a teratoma and the epithelial tubule, possibly a proximal tubule (white arrow). (J) Isotype-matched nega-
tive control for I. Scale bar = 100 μm.
DIFFERENTIATION OF HES CELLS TO PUTATIVE RENAL PROGENITORS
Enrichment of Human ES-derived renal cells
by FACS selection and characterization
by gene expression analysis
The majority of the 26 or more cell types of the adult
kidney arise primarily from the MM [ 28 ]. Therefore, the
differentiation strategy employed aimed to produce cells
of this phenotype. Reduction of serum concentration was
selected as it has previously been used to induce human
ES cell differentiation toward the pancreatic and cardio-
myocyte lineages [ 34 , 36 ], suggesting selection for mesendo-
dermal tissues. Furthermore, we consistently observe more
marked differentiation in colonies of human ES cells cul-
tured on a reduced density of feeder cells (unpublished).
However, inducing human ES cells to differentiate under
these culture conditions results in a heterogeneous popula-
tion of cells comprised of cells of interest interspersed with
a myriad of other cell types. We therefore sought to enrich
for renal cells using FACS selection. Two cell surface mark-
ers, CD24 and podocalyxin, were selected for FACS based
on microarray and in situ hybridization assays performed
by Challen and colleagues [ 30 ]. In the latter study, CD24
identifi cation of the rare putative regions of renal tissue,
serial sections were then stained with epithelial or renal
markers ( Fig. 1G—1J ). Structures reminiscent of renal
tubules and collecting ducts marked by cadherin 16 were
observed ( Fig. 1G ). WT1 was also localized to nonorga-
nized regions of the teratoma ( Fig. 1H ). Glomerular-like
structures were observed in the teratoma and displayed a
remarkable degree of organization as evidenced by Figure
1I and 1J . This glomerular-like structure stained positively
for podocalyxin and demonstrated a structure resembling
a renal corpuscle with a glomerulus (with podocytes), uri-
nary space, Bowman’s parietal epithelium, and a urinary
pole extending into the proximal tubule (white arrow).
The formation of glomerular-like structures immunoreac-
tive for the podocyte marker (in this context) podocalyxin
alongside the cadherin 16 immunoreactive tubule-like
structure provides strong evidence that human ES cells in
a teratoma are capable of forming organized kidney-like
structures. However, these structures were exceedingly
rare and their formation was time-consuming (2–3 months
duration). Therefore, in vitro strategies for the production
of renal cells were explored.
Normalised Gene Expression Relative
to Unfractionated Cells [Log (RQ)]
GCTM2 –veGCTM2 Lo GCTM2 Hi
150 200 250
FIG. 2. Cell-sorting strategy. (A)
Representative FACS plots showing vi-
able, differentiated human embryonic
stem (ES) cells fractionated based on
co-expression of CD24, podocalyxin,
and varying levels of GCTM2 (denoted
++Hi, ++Low, and ++−). (a) Cells
were fi rst distinguished based on for-
ward and side scatter and (b) dead cells
were excluded based on the uptake of
the viability marker propidium iodide.
(d) Cells that co-expressed CD24 and
podocalyxin were further gated based
on GCTM2 expression resulting in 3
populations. (B) Quantitative gene ex-
pression analysis indicates that when
compared with unfractionated cells, the
++Low and ++− fractions showed a
trend of increased expression of kidney
development genes LHX1, PAX2, and
WT1 and reduced expression of plurip-
otent genes OCT4 and GDF3. * indicates
a statistically signifi cant difference.
Values are presented as means ± stan-
dard error of the mean (SEM), n = 5.
LIN ET AL. 1642
While podocalyxin and GCTM2 have previously been
reported to be identical [ 43 ], we do not believe this to be
the case [ 41 ]. Live cells showing double-positive immuno-
reactivity to CD24 and podocalyxin were fractionated into
populations according to expression level of GCTM2 and
denoted ++Hi, ++Low, and ++−, respectively ( Fig. 2A ).
These fractions were then analyzed for gene expression by
Figure 2B shows that in differentiated cultures of the cell
line HES-4, expression of the pluripotency genes OCT4 and
GDF3 were down-regulated in both the ++Low and ++−
was strongly up-regulated in the uninduced murine MM
at E10.5 while podocalyxin was highly expressed in MM
and the surrounding intermediate mesoderm, which may
play a crucial role in directing nephrogenesis of the unin-
duced MM. These markers are not renal-specifi c and CD24
and podocalyxin transcripts were also detectable in highly
purifi ed human ES cell populations [ 41 , 42 ] and some differ-
entiated cell types. Therefore, a well-characterized marker
of pluripotency, GCTM2 [ 24 ], was included to discriminate
between undifferentiated (GCTM2 + ) and differentiated
(GCTM2 Low , GCTM2 − ) cells within the global population.
ES Derived Random
FIG. 3. Microarray analyses of undifferenti-
ated, unfractionated, and sorted fractions. (A)
Heat maps of undifferentiated, unfractionated,
and sorted fractions. Combined heat map of 3
biological replicates shows down-regulation
of stem cell genes (yellow) and up-regulation
of developmental genes (blue) across the un-
differentiated (UD), unfractionated (UF),
+Low, and ++− fractions. All 3 replicates
show similar expression patterns of pluripo-
tent and developmental genes confi rming the
reproducibility of the array results. (B) Gene
ontology analyses performed on the top 1,000
up-regulated genes (B-stat > 0) in the ++−
compared with the undifferentiated fraction
show a slight increase in percentage of kidney
and vascular development-associated genes
in the ++− fraction relative to either spon-
taneously differentiated embryonic stem (ES)
cells or cell populations derived from varying
stem cell marker expression-based fraction-
ation (red arrowheads). Ontology analysis
also indicated a reduced proportion of genes
involved in neural and skeletal muscle de-
velopment in the ++− cell fraction (black
arrowheads). (C) Cluster analyses. (a) Cluster
analysis of the top 200 up-regulated genes in
the ++− fraction compared with undifferen-
tiated ES cells cross-matched with a genito-
urinary development-specifi c gene expression
database (GUDMAP) shows an overrepresen-
tation of transcripts associated with E11.5
murine metanephric interstitium (green box)
and E15.5 nephrogenic and cortical intersti-
tium (yellow box). (b) This overrepresentation
was not observed when a randomly generated
list of genes was subjected to the same cross-
match. (c) Cluster analysis performed on all
genes up-regulated only in ++− compared
with undifferentiated, spontaneously differ-
entiated, and ++Low fractions shows these
genes are associated with the MM at E11.5
(green box), podocytes at E13.5 and E15.5 (yel-
low box), and the collecting duct and proximal
tubules (purple box) when cross-matched with
the GUDMAP database.
DIFFERENTIATION OF HES CELLS TO PUTATIVE RENAL PROGENITORS
urogenital development obtained from the Genitourinary
Development Molecular Anatomy Project (GUDMAP) [ 44 ].
Genes were clustered based on spatial and temporal expres-
sion within the kidney. Figure 3C shows that these up-regu-
lated genes cluster into a group of genes associated with the
MM at E11.5 (prior to nephron formation) and the cortical
and nephrogenic interstitium at E15.5 of murine renal devel-
opment. This suggests selection for a MM-like population of
cells. Conversely, this clustering pattern was not observed
when a randomly selected list of genes was compared with
the same gene expression database. As this comparison was
performed with cells of the ++− fraction and undifferenti-
ated ES cells, it is possible that this may be due to spontaneous
differentiation and not the result of enrichment by our sort-
ing strategy. Therefore, we further identifi ed genes selectively
up-regulated in the ++− fraction compared with the ++Low,
spontaneously differentiated (GCTM2 − /CD9 − ), and undiffer-
entiated ES fractions. This list of genes was then cross-matched
again with those from GUDMAP [ 44 ] ( Supplementary Table
1; Supplementary materials are available online at www.
liebertonline.com/scd ). Results from this second comparison
show that genes that are up-regulated only in the ++− fraction
can indeed be clustered into a group associated with the MM
at E11.5 ( Fig. 3C ). It is likely that this fi nding was not observed
in our initial gene ontology analysis as GO utilizes general
markers of kidney development and provides an overview
compared with GUDMAP that focuses specifi cally on dis-
crete stages of kidney development. This result both validates
the sorting and differentiation strategy and corroborates the
predicted markers [ 30 ]. Furthermore, there appears to be a
cluster of genes associated with podocytes at E13.5 and E15.5,
the collecting duct, and proximal tubules ( Fig. 3C ).
Validation of microarrays by RT-QPCR
RNA from the same samples used in the microarray anal-
yses were reverse-transcribed and analyzed by RT-QPCR for
the expression of genes involved in development of various
mesoderm compartments. Results of the quantitative PCR
are expressed on a logarithmic scale and show that genes
expressed in all 3 mesoderm compartments are up-regu-
lated in the 2-week differentiated cells compared with the
calibrator human ES cell line. Genes associated with the in-
termediate mesoderm were also slightly up-regulated in the
++Low and ++− fractions compared with both the unfrac-
tionated and ++Hi populations ( Fig. 4 ). Notably, this pattern
of increased expression in the ++Low and ++− fractions
was also observed for genes involved in lateral mesoderm de-
velopment to a more pronounced extent ( Fig. 4 ). Conversely,
transcript levels of paraxial mesoderm genes were detected
at lower levels than those of the other mesoderm compart-
ments and furthermore did not show much variation in ex-
pression between the 4 analyzed fractions ( Fig. 4 ).
Detection of WT1 and PAX2 co-expressing cells
by immunolocalization confi rms the presence
of renal cells in FACS-fractionated cells
While RT-QPCR and transcriptome analyses provided
evidence of the presence of MM-like cells in the sorted
fractions, these were performed on heterogeneous popula-
tions of cells. In order to exclude the possibility that the
observed up-regulation of kidney-related genes is due to
fractions compared with the unfractionated population.
Conversely, transcripts of PAX2 and WT1 were up-regulated
( P ≤ 0.05) in the ++− fraction. Expression of LHX1 was sig-
nifi cantly increased in the ++Low but not in the ++− frac-
tion. Similar trends in gene expression were also observed
when human ES cell populations were fractionated postin-
duction from 2 other human ES cell lines tested, HES-2 and
HES-3 ( Supplementary Fig. 1 ; Supplementary materials are
available online at www.liebertonline.com/scd).
Microarray analysis of FACS-separated fractions
show up-regulation of kidney-associated genes
in the differentiated populations
While we detected an up-regulation in the levels of
PAX2, WT1, and LHX1 in the differentiated fractions, we
recognize that these markers individually are not specifi c
for renal cells. As such, to further confi rm the presence of
potential renal progenitors in our differentiated human ES
cell population, we utilized microarray analysis to examine
the global gene expression profi le of the FACS-separated
fractions. RNA from 4 biological replicates of each fraction
was isolated and analyzed by Illumina microarray from the
following fractions of HES-4 cells ( n = 4 passages); unfrac-
tionated, ++Low , ++−, and undifferentiated ES cells of the
same passage. The gene expression patterns determined
by array analysis were then clustered according to their in-
volvement in pluripotency (yellow) or differentiation and
development (blue) and represented on a heat map ( Fig. 3A ).
The heat map shows that genes involved in development
are clearly up-regulated as the fractions move from undif-
ferentiated to unfractionated, ++Low, and ++− fractions,
while the reverse is true for stem cell genes. In addition,
results were subjected to gene ontology and cluster analysis
to characterize the fractions (full data sets are available at
GEO accession number GSE15257). Initially, the top 1,000
up-regulated genes in the ++− fraction relative to the un-
differentiated ES cells of the same passage were compared
with markers of development obtained from GO (www.
geneontology.org). To ensure that the bioinformatic readout
obtained was representative of the differentiation and isola-
tion regime, ontology analysis on the populations isolated
here was carried out in parallel with analysis of previously
obtained microarray results from spontaneously differen-
tiated human ES cell-derived populations [ 42 ]. The results
of the gene ontology analyses indicate a higher percentage
of renal development genes in the ++− fraction compared
with any other lineage investigated ( Fig. 3B ). While this in-
crease does not appear signifi cant when compared with the
spontaneously differentiated or GCTM2 − /CD9 − fractions, it
is interesting to note that genes associated with muscular
and neural development appear to be present at a lesser pro-
portion in the ++− fraction than either of the other 2 ana-
In order to determine if up-regulated transcripts in the
++− fraction are associated with a particular compart-
ment of the developing kidney or a specifi c time point of
kidney development, a bioinformatic cluster analysis was
performed. Expression levels of 200 of the most differen-
tially up-regulated genes in the ++− fraction relative to
the undifferentiated ES cells of the same passage were com-
pared with a transcriptome profi ling database specifi c to
LIN ET AL. 1644
increased gene expression in distinct cell populations con-
tained within the FACS-separated cell populations, we per-
formed indirect immunofl uorescent analysis of WT1 and
PAX2 expression on FACS-separated 2-week differentiated
human ES cells. To avoid cross-reactivity of the secondary
antibodies to monoclonal antibodies of identical isotypes,
differentiated human ES cells were sorted using GCTM2
alone instead of in combination with CD24 and podoca-
lyxin to yield GCTM2 − cells that contain the ++− cells of
interest before being labeled with WT1 and PAX2. A subset
of GCTM2 − /PAX2 + /WT1 + cells was detectable ( Fig. 5 ). In
addition, GCTM2 − cells that labeled positive for either
PAX2 or WT1 alone could also be seen in these fractionated
In this study, we demonstrate that it is possible to gen-
erate renal cells from human ES cells in vitro. Initially, we
established that the markers WT1, cadherin 16, and podo-
calyxin recognize structures derived from the MM and UB
in the developing human embryo. While the pluripotent na-
ture of human ES cells is well accepted, to date there have
been 2 reports of the formation of kidney-like structures
from human ES cell-derived teratomas [ 23 , 25 ]. Only one of
these reports presented immunohistochemistry for mark-
ers of renal structures (WT1 and NCAM) [ 25 ]. We have con-
fi rmed and extended these fi ndings using the renal markers
WT1, cadherin 16, and podocalyxin and show that human
ES cells indeed have the ability to differentiate into struc-
tures resembling fetal glomeruli. In addition, we also extend
these results to validate that the structures observed in the
teratomas are of the renal lineage.
In order to achieve the long-term goal of utilizing
human ES cells in cell replacement therapy for renal dis-
ease, methods for obtaining renal cells from human ES
cells in a controlled and reproducible manner are required.
To date, there has been only one study involving the di-
rected differentiation of human ES cells toward the renal
lineage, which indicated that it is possible to detect an up-
regulation of MM-related transcripts (OSR1, PAX2, SIX2,
and WT1) and genes associated with kidney precursors
( EYA1 , LIM1 , and CD24 ) in human ES cell colonies cultured
in the presence of retinoic acid, activin A, and BMP4 or
BMP7 on laminin or gelatin substrates [ 27 ]. In the present
study, we employed a strategy of serum concentration re-
duction and feeder depletion to induce differentiation of
human ES cells. As noted, serum reduction in combination
with growth factors such as activin A [ 35 , 36 ] or in a co-cul-
ture system [ 34 ] has previously been used to differentiate
human ES cells toward the cardiomyocyte and pancreatic
lineages, respectively. In the case of endoderm differenti-
ation, this effect at least partially relies on the reduction
of insulin-like growth factor (IGF), usually present in the
serum, which is thought to antagonize differentiation of
human ES cells via the activation of the phosphotidylinosi-
tol-3-kinase (PI3K) signaling pathway [ 45 ]. While the effect
of this pathway on differentiation toward other lineages
has not been investigated, we speculate that similar factors
are present in serum and either have an inhibitory effect
on differentiation, a stimulatory effect on the maintenance
of pluripotency, or both. As with most protocols involving
Normalised Gene Expression Levels
Normalised Gene Expression Levels
Normalised Gene Expression Levels
FIG. 4. Validation of microarray results by quantitative
polymerase chain reaction (PCR). Results of the microarray
were validated by quantitative PCR screen of genes involved
in the development of each compartment of the mesoderm
where expression levels were fi rst normalized against 18S
rRNA and calibrated using a CD30-expressing human em-
bryonic stem (ES) cell line as an internal standard. The QPCR
results demonstrated a slight up-regulation in transcript
levels of all 3 intermediate mesoderm and 2 lateral meso-
derm genes, CD34 and CDH5, in the ++− fraction compared
with the other 3 fractions, indicating that cells expressing
these transcripts are enriched from the unfractionated cell
population by sorting based on CD24+/Podo+/GCTM2−.
Conversely, an overall lower level of gene expression was
observed for paraxial mesoderm genes, which also showed
little variation in transcript abundance across the cellular
fractions, indicating that the cells that express these genes
are not selectively isolated by sorting with the above mark-
ers. Values are presented as means ± SEM, n = 3.
DIFFERENTIATION OF HES CELLS TO PUTATIVE RENAL PROGENITORS
differentiation based on their down-regulation of the stem-
ness genes, OCT4 and GDF3 . With particular regards to
HES-4, the observed up-regulation of LHX1 transcripts in
the ++Low but not the ++− fraction lends further weight
to the hypothesis that this fraction contains less differenti-
ated cells than ++− since Lim1 is critical for intermediate
mesoderm formation in the mouse. While the quantitative
PCR readout indicated that HES-4 is the cell line that is
most amenable to the differentiation regime, similar trends
in gene expression were observed in the other 2 human ES
cell lines ( Supplementary Table 1 ). This variation in re-
sponsiveness of different cell lines is not uncommon and
has been reported with differentiation protocols aimed at
obtaining pancreatic endocrine cells from human ES cells
[ 46 ]. Notably, these gene expression results are in line with
reports by D’Amour and colleagues (2005) who showed
that culture of human ES cells for 4 days in the presence of
low serum results in high expression of mesoderm genes.
As organogenesis is a complex process that involves
the expression of a multitude of genes, evidence of lineage
commitment requires larger-scale characterization via
human ES cell differentiation, the resulting cell population
represented a heterogeneous mixture of cell types. The
markers CD24, podocalyxin, and GCTM2 were selected for
use in FACS to isolate renal cells from this mixture of cells.
Individually, neither CD24 nor podocalyxin are restricted
in their expression to the developing kidney. However,
using them in combination with GCTM2 exclusion enables
a subset of differentiated cells containing the renal pre-
cursor cells of interest to be isolated from a global hetero-
geneous population of differentiated and pluripotent cells.
We therefore isolated 3 fractions of cells CD24 + /podoca-
lyxin + /GCTM2 Hi (++Hi), CD24 + /podocalyxin + /GCTM2 Low
(++Low), and CD24 + /podocalyxin + /GCTM2-(++−). While
each of these fractions are still heterogeneous, we hypoth-
esized that the ++Hi fraction would contain the highest
proportion of pluripotent cells, the ++Low fraction con-
tains cells that have begun to commit to a mesoderm fate,
while the ++− fraction contains the renal cells of interest.
We confi rmed this hypothesis using quantitative gene ex-
pression analysis and showed that the ++Low and ++−
fractions have indeed proceeded along the pathway of
FIG. 5. Detection of a pop-
ulation of WT1+/PAX2+ cells.
(A–D) Four fi elds of view
showing the presence of a
rare population of cells that
co-localize WT1 and PAX2
in GCTM2− cell fractions
(arrow). (E) Magnifi ed image
of a cell co-expressing WT1
and PAX2. (F) Corresponding
nuclear stain for the isotype
LIN ET AL. 1646
from the Australian National Health & Medical Research
Committee (ALL), Kidney Health Australia (ALL) and
from the National Institute for Diabetes, Digestion and
Kidney Disease, National Institutes of Health (DK63400
to SG, MHL, MFP, SDR, and JFB) as part of the Stem Cell
Genome Anatomy Project (www.scgap.org). SAL was sup-
ported by a postgraduate scholarship from Kidney Health
Author Disclosure Statement
No competing fi nancial interests exist.
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transcriptome profi ling. Our microarrays provided strong
evidence of a MM-like population within the FACS-isolated
fractions of differentiated human ES cells. From our gene
ontology results, the observed up-regulation of kidney-as-
sociated genes and the signifi cant decrease in transcripts
associated with neural and muscular development in the
++− fraction relative to spontaneously differentiated
human ES cells is particularly encouraging, as the induc-
tion of neural differentiation is a default pathway that
occurs autonomously when inhibitory signals are removed
[ 47 ]. With respect to muscular lineages, while it appears
that the sorting strategy may also be selecting against
cells that have differentiated along muscular lineages,
the ontology analysis for markers of muscle development
includes genes associated with skeletal, cardiac, visceral,
and smooth muscles based on cluster and gene ontology
analyses. The effi cacy of our sorting strategy is also prom-
ising based on results of our cluster analysis, which show
that the ++− fraction contains cells that have an increased
expression of genes associated with specifi c compartments
of the kidney during development.
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tion enriched in cells adopting a phenotype derived from
intermediate mesoderm, the source tissue for the kidney.
In addition, this fraction showed differential expression
of markers previously associated with the MM. As noted
previously, this tissue gives rise to both the nephrons and
components of the renal interstitium. The former target is
the most critical for any potential renal regenerative option.
There are many known transcription factors that differ-
entiate between the MM fated to become nephron versus
interstitium and while this differentiation and enrichment
strategy showed up-regulation of MM markers, this did
not include those specifi cally marking the nephron pro-
genitors. For the MM to undergo a mesenchymal to epithe-
lial transition to form a nephron, the cells must co-express
WT1 and PAX2. While expression of each of these markers
individually have been reported in a variety of nonrenal
cell types, their co-expression during embryonic develop-
ment is a strong indicator of nephron progenitor activity
[ 22 ]. We identifi ed a small population of WT1 + PAX2 + cells
within the ++− fraction suggesting that such progenitors
were being induced at a low rate. What will be critical now
is to optimize further selective strategies for the enrich-
ment or selective culture of that nephron progenitor sub-
fraction. Such a population would be invaluable as a source
for the terminal differentiation of mature renal cells from
human ES cells for toxicology screening and potentially for
The authors thank Dr. David Nikolic-Paterson for pro-
viding the podocalyxin antibody (PHM5), Genevieve
Browne and Stephanie Wood for technical assistance with
immunohistochemistry, the Human Embryonic Stem Cell
Core Facility at the Australian Stem Cell Centre (ASCC)
for provision of cells, and FlowCore (a collaborative ini-
tiative between Monash University, the ASCC, and the
Australian Regenerative Medicine Institute [ARMI]) for
assistance with FACS. This work was supported by grants
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Received for publication January 12, 2010
Accepted after revision February 9, 2010
Prepublished on Liebert Instant Online February 9, 2010
Address correspondence to:
Dr. Andrew L. Laslett
Molecular and Health Technologies
C/O Australian Stem Cell Centre
Building 75 (STRIP)
Clayton VIC 3800
E-mail : firstname.lastname@example.org