Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture
et al. (2014) Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-
Culture. PLoS ONE 9(1): e86372. doi:10.1371/journal.pone.0086372
Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture
Richard L. Gieseck III
Nicholas R. F. Hannan
Neil A. Hanley
Rosemary A. L. Drake
Grant W. W. Cameron
Thomas A. Wynn
Majlinda Lako, University of Newcastle upon Tyne, United Kingdom
Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.
Funding: This work was funded by an ERC starting grant (L.V.), the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V.,
N.R.F.H.), the NIH-Oxford Cambridge Scholars Program Fellowship (R.L.G.), the Evelyn trust (N.R.F.H.), and the EU grant InnovaLiv (R.L.G, R.B., and L.V.). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Co-authors Rosemary A. L. Drake and Grant W. W. Cameron are employed by TAP Biosystems. These authors were included for their
collaborative work and technical assistance with the RAFT system. RAFT reagents were donated to the authors by TAP Biosystems for use in the experiments
described within the manuscript. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials.
With uses in drug screening, toxicology studies, cell-based
therapies, and in vitro disease modeling, primary human
hepatocytes (PHHs) are in high demand. However, lack of sufficient
organ donors, poor longevity in vitro, and difficulties with
dedifferentiation [1,2] have led researchers to seek alternative
sources to bridge the gap between clinical demands and cell
availability. Hepatocyte-like cells generated from hIPSCs
(IPSCHeps) have shown great promise to satisfy this need by providing
an inexhaustible source of cells that mirror the genotype of the
donor. Nevertheless, current differentiation protocols and
culture conditions often produce cells with a fetal identity and
suboptimal functionality compared to that of PHHs.
Several studies have demonstrated that culturing isolated PHHs
in a 3D format averts many effects of dedifferentiation and can
partially reverse this process in cells that have been cultured for
short term in monolayer (2-Dimensional conditions) . Such 3D
cultures have been shown to return the function of several
xenobiotic metabolizing enzymes to in vivo levels [8,9], reestablish
cellular polarization and canalicular structure [9,10], and maintain
other liver specific functions such as albumin secretion, glycogen
synthesis, and lipid storage [7,9]. Additionally, it has been
established that the presence and maintenance of cell-cell junctions
is critical to preservation of the mature hepatic phenotype .
However, 3D culture systems currently available are often
unwieldy and overly complex, leading to poor reproducibility
and restricting use to a few labs with highly specialized equipment.
Such methods, often based upon embryoid body differentiation,
are not compatible with high throughput screening and remain
difficult to apply to IPSC-Heps, which require long term,
reproducible culture for functional differentiation and subsequent
application in research and industry.
Based on these findings, we hypothesized that the phenotypic
profile of IPSC-Heps could be shifted towards PHHs by
transferring IPSC-Heps, which were fully differentiated in 2D,
into a 3D culture system. Furthermore, we hypothesized that the
maintenance of cell-cell junctions during the transfer procedure
would be vital to the preservation and maturation of the hepatic
phenotype. To test this, we conducted a direct comparison of
IPSC-Heps cultured on traditional 2D tissue culture plastic and
within the Real Architecture for 3D Tissues (RAFT) system.
This 3D culture matrix is based upon the concept of
concentrating a cell-seeded collagen hydrogel by removing
interstitial fluid [12,13] and allows for easily reproducible, type-I
collagen based, 3D cultures in a 96-well format. A neutralized
collagen solution is mixed with cells and subsequently is heated to
induce fibrillogenesis and encapsulate the cells in situ (Figure
S1a). A biocompatible absorber is placed on top of the collagen
hydrogel in order to remove fluid and collapse the construct to
physiological collagen densities. The low level of variability
between wells and plates, and the ability to easily control cell
and matrix density to produce physiologically relevant constructs,
made the RAFT system an ideal choice over traditional collagen
sandwich models. The single component, defined nature of the
construct made the system superior to Matrigel and other
ECMcytokine mixtures, which often yield high batch to batch variations
and can confound differentiation procedures. Additionally, the
96well format and the lack of need for complex, specialized
equipment was perfect for high throughput analyses.
In order to analyze the effects of this 3D culture system on
IPSC-Hep maturation, three IPSC lines were differentiated for
25 days towards the hepatic lineage using a common 2-D
differentiation protocol (Figure S1) . At this time, cells were
split into three sample groups and further differentiated for 10 or
20 days. Sample groups consisted of: 1) 2D control; 2) 3D culture
in which the cells were transferred to the RAFT matrix as small
epithelial clumps with cell-cell junctions intact (Figure S1b/c); 3)
3D culture in which the cells were completely dissociated,
disrupting the existing cell-cell junctions before transfer to the
RAFT matrix (Figure S1c). The three sample groups allowed us
to simultaneously probe the effects of 3D culture, maintenance of
cell-cell junctions, and culture time on the maturation of
Materials and Methods
Human iPS cell derivation and culture: Ethics for the iPSC lines
used in this study were approved under Addenbrookes Hospital
reference no. 08/H0311/201; R&D no. A091485. Additional
information can be found elsewhere . Adult Hepatocytes: Liver
samples were obtained in agreement with the rules of the hospitals
(Hospital La Fe, Valencia) ethics committee (CEIC, Comite Etico
de Investigacio n Clnica; approval number 2009/00111). Fetal
Hepatocytes: Human fetal tissue sample collection was approved
by NorthWest Ethics Committee (13/NW/0205). Additional
information can be found elsewhere . Written informed
consent from the donor or the next of kin was obtained for use of
all samples used within this study.
Tissue culture plastic (Corning) coated with porcine gelatin
(1 g/L; sigma) dissolved in water for embryo transfer (Sigma) for
30 minutes was pre-conditioned with MEF medium consisting of
Advanced DMEM/F-12 (Invitrogen), 10% FBS (Biosera), 1%
200 mM L-glutamine (Invitrogen), 1% penicillin/streptomycin
(10,000 U/mL; Invitrogen), and 0.0007% b-mercaptoethanol
(Sigma) for at least 12 hours prior to plating IPSC colonies.
hIPSCs were maintained feeder-free at 37uC, 5% CO2, 20% O2 in
chemically-defined, serum-free IPSC maintenance medium
(CDM-PVA) consisting 0.5 g of PVA (Sigma) dissolved in
250 mL of DMEM/F-12, GlutaMAX (Invitrogen), 250 mL of
IMDM (Invitrogen), 5 mL of chemically defined lipid concentrate
(Invitrogen), 20 mL of thioglycerol $97% (Sigma), 350 mL of
insulin (10 mg/mL; Roche), 250 mL of transferrin (30 mg/mL;
Roche) and 5 mL of penicillin/streptomycin (10,000 U/mL;
Invitrogen) supplemented with Activin A (10 ng/mL; R&D) and
FGF2 (12 ng/mL; R&D) . Media was changed daily and cells
were passaged every 57 days using a 1:1 mixture of collagenase
IV (400 mL of Advanced DMEM/F-12, 100 mL of KnockOut
Serum Replacement (Invitrogen), 5 mL of 200 mM L-glutamine,
3.5 mL of b-mercaptoethanol (14.3 M), and 500 mg of collagenase
type IV (Invitrogen)) and dispase II (500 mg of dispase II
(Invitrogen) dissolved in 500 ml of Advanced DMEM/F12) .
The three lines utilized in this study were BBHX8 , Line-B7
[4,5] (Referred to as BOB7 RM in this study), Line-B5 
(Referred to as BOB5 SC in this study).
2D common progenitor. IPSC lines were split (day 0) and
maintained for 48 hrs in CDM-PVA supplemented with Activin A
and FGF2 (media was changed daily for all subsequent steps, and
cells were differentiated at 37uC, 5% CO2, 5% O2, unless stated
otherwise). On days 23, cells were differentiated in CDM-PVA
supplemented with Activin A (100 ng/mL), FGF2 (80 ng/mL),
BMP4 (10 ng/mL; R&D), 10 mM LY-294002 (Promega), and
3 mM Stemolecule CHIR99021 (StemGent). On day 4, cells were
differentiated in CDM-PVA supplemented with Activin A
(100 ng/mL), FGF2 (80 ng/mL), BMP4 (10 ng/mL; R&D), and
10 mM LY-294002. On day 5, cells were differentiated in RPMI
Medium (RPMI 1640 Medium, GlutaMAX (Invitrogen), 2% B-27
Serum-Free Supplement (50X) (Invitrogen), 1% MEM
NonEssential Amino Acids Solution (100X) (Invitrogen), 1%
penicillin/streptomycin) supplemented with Activin A (100 ng/mL) and
FGF2 (80 ng/mL). On day 6, cells were expanded in RPMI
medium supplemented with Activin A (50 ng/mL). On day 7, cells
were split using Cell Dissociation Buffer (Enzyme-free, Hanks;
Invitrogen) and were plated in gelatin-coated, MEF media
conditioned 6-well plates at a density of 105,000 cells/cm2 in
RPMI+Activin A (50 ng/mL)+Y-27632 2HCl (10 mM
Selleckchem) . Cells were maintained in RPMI+Activin A (50 ng/
mL) on days 89. From day 10 onward, cells were matured in
Hepatozyme-SFM (Invitrogen) supplemented with 1% 200 mM
L-glutamine, 1% penicillin/streptomycin, 2% MEM
Non-Essential Amino Acids Solution (100X), 2% chemically defined lipid
concentrate, 0.14% insulin, 0.28% transferrin, hepatocyte growth
factor (50 ng/mL, Peprotech), and oncostatin M (10 ng/mL,
R&D) with media changed every other day.
3D-Single cell culture. Cultures designated for 3D single cell
culture followed the 2D common progenitor protocol described
above until day 25. At day 25, media was removed, wells were
washed with DPBS (Invitrogen), and 1 mL of Cell Dissociation
Buffer pre-warmed to 37uC was placed in each well. The plates
were incubated at 37uC, 5% CO2, 5% O2 for 15 minutes (at which
time half of the wells were subjected to the clump culture protocol
below) or 45 minutes, until cells dispersed as single cells. Cells were
pelleted and washed twice with Hepatozyme-SFM. Cells were
counted and resuspended in Hepatozyme-SFM at a density of
1.396107 cells/mL for use in the RAFT system (RAFT Standard
Protocol available online; TAP Biosystems). Cells embedded
within 3D cultures were maintained in
Hepatozyme-SFM+supplements with media changes every other day.
3D-Clump culture. Cultures designated for 3D clump
culture followed the 2D common progenitor protocol and 3D
single cell protocol above until the 15-minute dissociation step. At
this point, cells were removed from the surface in clumps using
manual perturbation with a 5 mL serological pipette tip. Cells
were pelleted and washed twice with Hepatozyme-SFM. Cell
count was estimated using the count from the single cells, and cells
were resuspended in Hepatozyme-SFM at a density of 1.396107
cells/mL for use in the RAFT system (RAFT Standard Protocol
available online; TAP Biosystems). Cells embedded within 3D
cultures were maintained in Hepatozyme-SFM+supplements with
media changes every other day.
Primary Human Controls
Adult hepatocytes. Liver samples were obtained in
agreement with the rules of the hospitals ethics committee. None of the
donors (4 men aged between 54 and 80) were regular consumers of
alcohol or of other drugs and were not suspected of harboring any
infectious disease. Human hepatocytes were isolated from liver
biopsies (,5 g) using a two-step collagenase perfusion technique.
Hepatocytes were seeded and cultured as previously described in
Fetal hepatocytes. RNA isolated from human fetal liver
samples relating to an approximate gestational age of 7.5 weeks
was generously donated by Drs. Andrew Berry and Neil Hanley of
the University of Manchester.
Cells were fixed for 30 minutes at 4uC in 4% paraformaldehyde
(PFA) and washed 3 times with DPBS. Cells were blocked for
1 hour with DPBS containing 1% donkey serum (Serotec Ltd.),
1% Triton X-100 (Sigma). Cells were incubated for 1 hour at
room temperature with the following primary antibodies diluted in
the blocking solution: A1AT (1:100; DAKO, cat. no. A0012), AFP
(1:100; DAKO, cat. no. A0008), ALB (1:100; DAKO, cat. no.
A0008), ASGPR (1:100; Thermo Scientific, cat. No. MA1-40244),
b-Catenin (1:100; Abcam, cat. No. ab32572 ), CD26 (1:100;
Abcam, cat. no. ab28340), CK18 (1:100; SantaCruz, cat. no.
sc6259), HNF4 (1:100; SantaCruz, cat. no. sc6556), Ki-67 (1:100;
Abcam, cat. no. ab15580), MRP2 (1:100; Abcam, cat. no.
ab3373). Cells were washed three times with PBS for 30 minutes
each. Cells were incubated for 1 hour at room temperature with
appropriate secondary antibodies diluted in the blocking solution:
Alexa Fluor 488 Series (1:1000, Invitrogen, cat. nos. A-11055/
21202/21206) and Alexa Fluor 568 Series (1:1000, Invitrogen, cat.
nos. A-10037/10042/11057). Nuclei were stained using
bisbenzimide (1:10,000 in DPBS; sigma) for 30 minutes. Cells were then
washed three times with PBS for 30 minutes each and then imaged
using an LSM700 laser scanning confocal microscope (Carl Zeiss).
Periodic Acid Staining
Fixed samples were triple rinsed with deionized water and then
placed in 0.5% periodic acid solution (Thermo Scientific) for 5
minutes at room temperature. Samples were then rinsed with
deionized water for 5 minutes before being submerged in Schiff
Reagent (Thermo Scientific) for 15 minutes. Samples were rinsed
with lukewarm tap water for 10 minutes. Samples were then
counterstained with Hematoxylin I (Thermo Scientific) for 1
minute. Samples were rinsed with deionized water for 30 seconds
and placed in 12 mM sodium bicarbonate for 1 minute. Samples
were rinsed once with deionized water and then twice with 100%
ethanol for 1 minute each. Samples were triple rinsed for one
minute each with 120 mM hydrochloric acid in 70% ethanol
Oil Red O Staining
Fixed samples were washed with tap water for 5 minutes.
Samples were then rinsed with 60% isopropanol for 5 minutes.
30 mL Oil Red O Solution (0.5 g Oil Red O (Sigma) in 100 mL
isopropanol) was mixed with 20 mL distilled water and filtered
using a 0.24 mm vacuum filter to making a working solution.
Samples were submerged in the working solution for 15 minutes
and then rinsed with 60% isopropanol. Samples were
counterstained with Hematoxylin I (Thermo Scientific) for 1 minute.
Samples were rinsed with deionized water for 30 seconds and
placed in 12 mM sodium bicarbonate for 1 minute. Samples were
triple rinsed with DPBS before imaging.
Scanning Electron Microscopy
Sample preparation. Incisions through areas of interest
within PFA fixed 3D cultures were made manually using a scalpel
and bright field microscope. Sections of interest were fixed with
1.5% glutaraldehyde (EM-grade; Sigma) in 1 M sodium
cadodylate (Sigma) for 2 hours. Sections were rinsed with 50%, 90% and
100% ethanol for 5 min, 5 min, and 15 min respectively. The
sample was saturated with hexamethyldisilazane (Sigma) three
times for 3 minutes each and then dried overnight in a chemical
safety cabinet. Samples were mounted using double-sided carbon
tape using minimal force to ensure adhesion. An SC7640 sputter
coater (Polaron) was used to coat the samples with Au for 90
Quantitative RT-PCR (qPCR)
Total RNA was extracted using GenElute Mammalian Total
RNA Miniprep Kit (Sigma). 500 ng of total RNA were
reversetranscribed using Superscript II Reverse Transcriptase
(Invitrogen). qPCR reaction mixtures were prepared as described
(SensiMix SYBR Low-ROX Kit protocol; Bioline). The mixture
was denatured at 95uC for 10 minutes, cycled 40 times (95uC for
30 seconds, 60uC for 30 seconds, 72uC for 30 seconds), followed by
final extension at 72uC for 10 minutes. Primer templates can be
found in the Supplementary Information. qPCR reactions were
performed using a Stratagene Mx3005P in technical duplicates
and biological triplicates. All genes were normalized to the
geometric mean of PBDG, RPLP0, GAPDH, and HDAC and
were normalized to the expression of undifferentiated IPSCs using
the DDCt method unless stated otherwise. HDAC was utilized due
to its very stable expression upon liver differentiation. This was
validated by several control experiments comparing HDAC
expression to other common housekeeping genes (data not
shown). Primer sequences used in this study can be found in
Table S1. Statistical significance is shown in Tables S2S7.
Cytochrome P450 Activity
CYP3A4 activity for the 10 day intervals was measured using
P450-Glo Assays (Promega). Cells were incubated with 3 mM
luciferin-IPA in Hepatozyme-SFM for 60 minutes prior to media
collection. Luminescence was measured using a GloMax 96
Microplate Luminometer (Dual Injectors; Promega) using the
built-in P450-Glo acquisition protocol. CYP3A4 Activity for the
D35 2D sample was also assessed by the rate of conversion of
Midazolam to 19-HO-Midazolam using HPLC-MS. HPLC-MS
analysis was performed only on one sample group (2D day 35,
Figure S10) since this analysis requires complete sacrifice of the
culture. This sample was analysed first using the P450-Glo assay
and then using HPLC-MS, serving as a way to link the two
methods of measuring CYP3A4. This allowed us to compare the
functionality of our time course experiment (completed using the
P450-Glo assay) to the 35 adult primary samples, which were
analysed using the HPLC-MS analysis through the InnovaLiv
project, without having to sacrifice all groups for this analysis.
Protein Quantification Assays
All protein quantification assays were performed by the
Cambridge Biomedical Research Centre Core Biochemical Assay
Laboratory following the protocols listed below.
Alpha-1-antitrypsin DELFIA. A1AT was measured using a
time-resolved fluorescence immunoassay on the DELFIA assay
platform. Nunc MaxiSorp plates were coated with rabbit
antihuman A1AT polyclonal antibody (Siemens) diluted in
bicarbonate coating buffer. The plate was incubated overnight and washed
four times with DELFIA wash buffer (PerkinElmer) before
blocking with 300 mL of 1% BSA in PBS for 1 hour. The plate
was washed four more times with DELFIA wash buffer before use.
The assay was calibrated with a human serum standard (Siemens).
The standard was serial diluted in DELFIA multibuffer
(PerkinElmer) to produce 9 standards with a concentration range of 500
to 3.9 ng/mL. Multibuffer was used as the zero concentration
standard. 90 mL of multibuffer was added to each well of the plate
followed by 10 mL of standard or unknown sample in technical
duplicate. The plate was sealed with a plate sealer and incubated
on a plate shaker for 2 hrs at room temperature. The plate was
then washed four times with wash buffer and 100 mL of
biotinylated goat anti-human AAT polyclonal antibody diluted
in multibuffer was added to the plate. The plate was sealed with a
plate sealer and incubated on a plate shaker for 2 hrs at room
temperature. The plate was then washed four times with wash
buffer and 100 mL of streptavidin-europium conjugate
(PerkinElmer) diluted in multibuffer was added to the plate. The plate was
sealed with a plate sealer and incubated on a plate shaker for 30
minutes at room temperature. The plate was then washed six times
with wash buffer and 200 mL of enhancement solution
(PerkinElmer) was added to the plate. The plate was incubated on a plate
shaker for 5 minutes followed by 5 minutes on the bench before
reading time-resolved fluorescence in the Victor3 plate reader
(PerkinElmer). Results were calculated using the PerkinElmer
MultiCalc software package.
immunoassay. Albumin was measured using the MesoScale
Discovery assay platform. Mesoscale standard bind plates were
coated with a goat anti-human albumin polyclonal antibody
(Bethyl laboratories) diluted in PBS. The plate was incubated
overnight and washed three times with PBS/Tween wash buffer
before use. The assay was calibrated with a human serum
preparation (Multiqual; Biorad). The preparation was diluted in
MSD Diluent 7 (MesoScale Discovery) to produce a series of 8
standards with a concentration range of 1000 to 15.6 mg/L. MSD
Diluent 7 was used as the zero concentration standard. 30 mL of
MSD Diluent 7 was added to each well of the plate followed by
10 mL of standard or unknown sample in technical duplicate. The
plate was sealed with a plate sealer and incubated on a plate
shaker for 2 hrs at room temperature. The plate was then washed
three times with PBS/Tween wash buffer and 25 mL of rabbit
anti-human albumin polyclonal antibody diluted in MSD Diluent
100 was added to the plate. The plate was sealed with a plate
sealer and incubated on a plate shaker for 1 hr at room
temperature. The plate was then washed three times with PBS/
Tween wash buffer and 25 mL of goat anti-rabbit
IgG-SulphoTAG diluted in MSD Diluent 100 was added to the plate. The
plate wa sealed with a plate sealer and incubated on a plate shaker
for 30 minutes at room temperature. The plate was then washed
three times with PBS/Tween wash buffer and 150 mL of 1X Read
Buffer T (MesoScale Discovery) was added to the plate. The plate
was read immediately on a SECTOR Imager 6000. Results are
calculated using the MSD Discovery Workbench software
Alpha-fetoprotein DELFIA. AFP was quantified using the
commercially available DELFIA hAFP kit and protocol
Standard error measurements and sample means were
calculated for all conditions and subjected to unpaired, two-tailed,
Welchs t-tests. P-values below 0.05 were considered significant for
this study. Hierarchical clustering was performed using Euclidean
distances with unweighted pair-group methods using centroids.
Calculation of Average Global Change in Fold Expression
Average change of a culture condition in fold expression for the
39 genes analyzed in aggregate compared to the 2D Progenitor
Culture was calculated according to the following formula:
Average Change in Expression
P39 Fold Expression of Gene n f or Culture Condition
~ n~1 Fold Expression of Gene n for 2D Progenitor Culture
Results and Discussion
Cell-cell Junctions are Necessary for the Maintenance of
the Hepatic Phenotype in 3D
We began by investigating the importance of cell-cell junction
maintenance during the transfer of the cells from 2D to 3D culture
(Figure 1). Media samples were taken at day 25, 35, and 45 and
were subjected to immunoassays in order to quantify the secretion
of human serum albumin, alpha-1-antitrypsin (A1AT), and
alphafetoprotein (AFP), which marks specifically fetal hepatocytes. At
day 45, 3D clump cultures demonstrated a 10-fold increase in
albumin secretion, a 1.5-fold increase in A1AT secretion, and a
20-fold decrease in AFP secretion compared to the day 25
common progenitor (Figure 1a). Conversely, 3D single cell
cultures demonstrated a 10-fold decrease in albumin secretion and
a complete loss of detectable A1AT. Furthermore, AFP secretion
decreased by 1500-fold in single cells suggesting a general decline
in hepatic phenotype. Increasing the density of single cell cultures
to mimic the local cell density within clumps had no significant
effect on the phenotype (data not shown). Together these data
show that cell-cell junction maintenance is necessary for
HepsIPSC differentiation and that 3D culture could accelerate the
decrease of fetal markers such as AFP.
To confirm these observations, we compared the maturation
and hepatic phenotypic profile of IPSC-Heps to that of freshly
isolated adult hepatocytes by qPCR analyses of 39 hepatic genes,
including multiple phase I/II/III metabolic enzymes along with
several hepatic nuclear receptors. Hierarchical clustering of the
profiles shows a distinct divergence in the two culture conditions
that increases with time. By day 45, the 3D single cell condition
lost detectable expression of 23 out of the 39 genes analyzed, with
an average gene expression of 0.0001% of primary adult
hepatocytes, nearly an 8,000-fold decrease from the day 25
common progenitor (Figures 1b, S2ac, S3ac, S4ac, S5). In
contrast, the 3D clump culture experienced an average 6-fold
increase compared to the progenitor and expressed all 39 genes
analyzed to varying degrees. Finally, localization and homogeneity
of protein expression (AAT, ALB, b-CAT, MRP2 and CD26)
were assessed by immunofluorescence. 3D clump cultures
demonstrated a homogenous population of cells with protein
expression and localization similar to that seen in PHHs, whereas
3D single cell cultures demonstrated significant heterogeneity in
expression (Figures 1c, S7, S8). Additionally, significant
Figure 1. Functional and transcriptional comparison of IPSC-Hep 3D cultures plated as single cells or clumps. (A) Secreted albumin,
alpha-fetoprotein, and alpha-1-antitrypsin levels as evaluated by immunoassays (mean 6 s.d.; n = 3 biological replicates). (B) qPCR heatmap of 39
hepatic genes comparing the two 3D culture conditions to adult and fetal hepatocytes (range of expression shown as sample extrema for each gene;
quantitative values shown in Figures S2S4). (C) Confocal micrograph highlighting the loss of detectable albumin in 3D single cell cultures and the
spontaneous polarization of IPSC-Heps within 3D clump cultures (scalebar = 100 microns).
irregularities in cell size and nucleus morphology along with
membrane blebbing were seen in the single cell cultures. This is
suggestive of contact-dependent apoptosis similar to that seen in
low density PHH dedifferentiation [1,2].
3D Clump Cultures Induce a more Mature Phenotype
Compared to 2D
Having confirmed the necessity of cell-cell junctions in
phenotypic maintenance, we conducted a direct comparison of
the 3D clump culture to 2D controls in order to determine the
functional benefits which 3D culture could confer. We began with
oil red o and periodic acid staining to determine differences in lipid
storage and glycogen synthesis respectively (Figure 2a). Both
cultures demonstrated the ability to store lipids and synthesize
glycogen; however, the 3D clump culture demonstrated a
significantly higher percentage of cells actively synthesizing
glycogen (.95% compared to 46% in 2D).
Furthermore, qPCR analysis using the 39 gene panel described
above demonstrated significant maturation events in Phase I/II/
III enzymes in addition to other hepatocyte associated genes
(Figures 2b, S2ac, S3ac, S4ac, S5). AFP and CYP3A7, both
markers of fetal hepatocytes [17,18], were decreased 20-fold and
140-fold respectively in the day 45 3D clump culture compared to
the day 45 2D control (Figure 2b). MAOB (Phase I), UGT1A1
(Phase II), NNMT (Phase II), and ABCC2 (Phase III) were increased
2.5-fold, 10-fold, 3.7-fold, and 7.3-fold respectively (Figure 2b).
The induction of ABCC2, a marker of hepatocyte polarity found
on the apical pole and bile canalicular surfaces , led us to
investigate cell polarity further. Immunostaining demonstrated the
presence of extensive canalicular formation throughout the 3D
clump cultures (as demonstrated by ABCC2 and CD26 ) and
an absence within the 2D controls. (Figures 1c, 2c, S6S9,
Videos S1S4). The establishment and maintenance of
IPSCHep polarity in 3D culture mediated through integrin-matrix
interactions is consistent with previous findings with primary
hepatocytes and has been shown to significantly decrease
dedifferentiation and increase longevity in these cells [1,2,21,22].
In order to assess any changes in functional longevity associated
with the 3D system, CYP3A4 activity was measured every 10 days
throughout the study using luciferase-based assays. No significant
differences were found in CYP3A4 activity between the two
culture conditions at day 35 or 45 (Figure 2d). However, between
days 45 and 55, cells in 2D culture consistently formed large
vacuoles and subsequently detached from culture surface. In
contrast, cells within the 3D matrix maintained levels of CYP3A4
activity at approximately 25% of that of PHHs (Figures 2D and
S10; n = 35 primary samples; range 10%200% activity of
individual primary samples; interpolated from HPLC-MS to
P450-Glo) from day 45 through 75. Although no further
maturation was observed during this period, we observed no
significant loss in CYP3A4 activity, demonstrating that the RAFT
system is conducive with long-term maintenance of cytochrome
activity. Our analysis ceased at day 75; however, cells could
potentially maintain functionality for even longer periods, making
this method ideal for long-term experiments needed for
physiologically relevant toxicology studies.
In summary, we have presented a method to easily improve the
maturation of current IPSC-Hep lines simply by transferring
existing cells as epithelial clumps into 3D collagen matrices. We
Figure 2. Functional comparison of IPSC-Hep 3D clump culture versus traditional 2D culture. (A) Oil red O and periodic acid staining
demonstrating lipid storage and glycogen synthesis in both 2D and 3D clump cultures. (B) qPCR analysis of select phase I and phase II enzymes,
hepatic transporters, and other hepatic markers demonstrating a shift towards a more mature phenotype in the 3D clump cultures (fold expression to
undifferentiated IPSCs; mean 6 s.d.; n = 3 biological replicates). (C) Confocal micrographs comparing the presence and localization of hepatic markers
within the two culture systems (scale bar = 100 microns). (D) CYP3A4 activity of the two culture conditions measured over a period of 75 days (mean
6 s.d.; n = 3 biological replicates).
have demonstrated that transition to 3D culture while maintaining
cell-junctions significantly shifts cell phenotype towards that of
primary hepatocytes compared to traditional 2D culture.
Additionally, 3D clump culture induces polarization and bile canaliculi
formation and extends the functional lifetime of the cells to over 75
days. Although further development is needed to generate fully
functional cells, our work represents a significant step for the
development of 3D systems for modeling liver diseases and testing
the toxic effects of various xenobiotics and suggests that this
method may be widely applicable to increase IPSC-Hep maturity.
Figure S1 Method to differentiate IPSC-Hep in 3D. (a)
Schematic of the RAFT process used in the maturation of
IPSCHeps. (b) Scanning electron micrograph of 3D clump culture
(scalebar = 5 microns). (c) Outline of the experiment used to probe
the effects of the three culture conditions on the maturation of
Figure S4 qPCR analysis for BBHX8. (ac) Fold expression
to undifferentiated IPSCs; mean 6 s.d.; n = 3 biological replicates.
Figure S6 Canalicular structures. Orthogonal analysis of
3D clump cultures demonstrating the presence of canalicular buds
(green ASGPR, Red HNF4a, blue Hoechst).
Figure S10 Cytochrome P450 Activity. CYP3A4 activity of
the 2D progenitor as assessed by the rate of conversion of
Midazolam to 19-HO-Midazolam using HPLC-MS (n = 35
List of primer sequences used for qPCR analyses.
Significance of BBHX8 qPCR analyses by Welchs
TSignificance of BOB5 SC qPCR analyses by Welchs
Significance of BOB7 RM qPCR analyses by Welchs
The authors would like to thank Dr. Cecile Villemant of TAP Biosystems
for her assistance with SEM imaging. We thank the Cambridge Biomedical
Research Centre Core Biochemical Assay Laboratory for undertaking the
human albumin, AFP, and A1AT protein quantifications. We would like to
thank the Unidad de Transplante Hepatico, Hospital La Fe, Valencia for
supplying liver biopsies and Unidad de Hepatologa Experimental, IIS La
Fe, Valencia for human hepatocyte isolation.
Conceived and designed the experiments: RLG NRFH TAW LV.
Performed the experiments: RLG RB. Analyzed the data: RLG.
Contributed reagents/materials/analysis tools: NH RALD GWWC RB.
Wrote the paper: RLG.
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